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Sample records for knock-out mice show

  1. Bex1 knock out mice show altered skeletal muscle regeneration

    PubMed Central

    Koo, Jae Hyung; Smiley, Mark A.; Lovering, Richard M.; Margolis, Frank L.

    2008-01-01

    Bex1 and Calmodulin (CaM) are upregulated during skeletal muscle regeneration. We confirm this finding and demonstrate the novel finding that they interact in a calcium-dependent manner. To study the role of Bex1 and its interaction with CaM in skeletal muscle regeneration, we generated Bex1 knock out (Bex1-KO) mice. These mice appeared to develop normally and are fertile, but displayed a functional deficit in exercise performance compared to wild type (WT) mice. After intramuscular injection of cardiotoxin, which causes extensive and reproducible myotrauma followed by recovery, regenerating muscles of Bex1-KO mice exhibited elevated and prolonged cell proliferation, as well as delayed cell differentiation, compared to WT mice. Thus, our results provide the first evidence that Bex1-KO mice show altered muscle regeneration, and allow us to propose that the interaction of Bex1 with Ca2+/CaM may be involved in skeletal muscle regeneration. PMID:17884015

  2. Bex1 knock out mice show altered skeletal muscle regeneration

    SciTech Connect

    Koo, Jae Hyung Smiley, Mark A.; Lovering, Richard M.; Margolis, Frank L.

    2007-11-16

    Bex1 and Calmodulin (CaM) are upregulated during skeletal muscle regeneration. We confirm this finding and demonstrate the novel finding that they interact in a calcium-dependent manner. To study the role of Bex1 and its interaction with CaM in skeletal muscle regeneration, we generated Bex1 knock out (Bex1-KO) mice. These mice appeared to develop normally and are fertile, but displayed a functional deficit in exercise performance compared to wild type (WT) mice. After intramuscular injection of cardiotoxin, which causes extensive and reproducible myotrauma followed by recovery, regenerating muscles of Bex1-KO mice exhibited elevated and prolonged cell proliferation, as well as delayed cell differentiation, compared to WT mice. Thus, our results provide the first evidence that Bex1-KO mice show altered muscle regeneration, and allow us to propose that the interaction of Bex1 with Ca{sup 2+}/CaM may be involved in skeletal muscle regeneration.

  3. Accelerated retinal aging in PACAP knock-out mice.

    PubMed

    Kovács-Valasek, Andrea; Szabadfi, Krisztina; Dénes, Viktória; Szalontai, Bálint; Tamás, Andrea; Kiss, Péter; Szabó, Aliz; Setalo, Gyorgy; Reglődi, Dóra; Gábriel, Robert

    2017-02-13

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.

  4. IL-4 Knock out Mice Display Anxiety-like Behavior

    PubMed Central

    Moon, Morgan L.; Joesting, Jennifer J.; Blevins, Neil A.; Lawson, Marcus A.; Gainey, Stephen J.; Towers, Albert E.; McNeil, Leslie K.; Freund, Gregory G.

    2015-01-01

    Inflammation is a recognized antecedent and coincident factor when examining the biology of anxiety. Little is known, however, about how reductions in endogenous anti-inflammatory mediators impact anxiety. Therefore, mood- cognition- and anxiety-associated/like behaviors were examined in IL-4 knock out (KO) mice and wild-type (WT) mice. In comparison to WT mice, IL-4 KO mice demonstrated decreased burrowing and increased social exploration. No differences were seen in forced swim or saccharine preference testing. IL-4 KO mice had similar performance to WT mice in the Morris water maze and during object location and novel object recognition. In the elevated zero-maze, IL-4 KO mice, in comparison to WT mice, demonstrated anxiety-like behavior. Anxiety-like behavior in IL-4 KO mice was not observed, however, during open-field testing. Taken together, these data indicate that IL-4 KO mice display state, but not trait, anxiety suggesting that reductions in endogenous anti-inflammatory bioactives can engender subtypes of anxiety. PMID:25772794

  5. IL-4 Knock Out Mice Display Anxiety-Like Behavior.

    PubMed

    Moon, Morgan L; Joesting, Jennifer J; Blevins, Neil A; Lawson, Marcus A; Gainey, Stephen J; Towers, Albert E; McNeil, Leslie K; Freund, Gregory G

    2015-07-01

    Inflammation is a recognized antecedent and coincident factor when examining the biology of anxiety. Little is known, however, about how reductions in endogenous anti-inflammatory mediators impact anxiety. Therefore, mood- cognition- and anxiety-associated/like behaviors were examined in IL-4 knock out (KO) mice and wild-type (WT) mice. In comparison to WT mice, IL-4 KO mice demonstrated decreased burrowing and increased social exploration. No differences were seen in forced swim or saccharine preference testing. IL-4 KO mice had similar performance to WT mice in the Morris water maze and during object location and novel object recognition. In the elevated zero-maze, IL-4 KO mice, in comparison to WT mice, demonstrated anxiety-like behavior. Anxiety-like behavior in IL-4 KO mice was not observed, however, during open-field testing. Taken together, these data indicate that IL-4 KO mice display state, but not trait, anxiety suggesting that reductions in endogenous anti-inflammatory bioactives can engender subtypes of anxiety.

  6. Prion Protein (PrP) Knock-Out Mice Show Altered Iron Metabolism: A Functional Role for PrP in Iron Uptake and Transport

    PubMed Central

    Singh, Ajay; Kong, Qingzhong; Luo, Xiu; Petersen, Robert B.; Meyerson, Howard; Singh, Neena

    2009-01-01

    Despite overwhelming evidence implicating the prion protein (PrP) in prion disease pathogenesis, the normal function of this cell surface glycoprotein remains unclear. In previous reports we demonstrated that PrP mediates cellular iron uptake and transport, and aggregation of PrP to the disease causing PrP-scrapie (PrPSc) form results in imbalance of iron homeostasis in prion disease affected human and animal brains. Here, we show that selective deletion of PrP in transgenic mice (PrPKO) alters systemic iron homeostasis as reflected in hematological parameters and levels of total iron and iron regulatory proteins in the plasma, liver, spleen, and brain of PrPKO mice relative to matched wild type controls. Introduction of radiolabeled iron (59FeCl3) to Wt and PrPKO mice by gastric gavage reveals inefficient transport of 59Fe from the duodenum to the blood stream, an early abortive spike of erythropoiesis in the long bones and spleen, and eventual decreased 59Fe content in red blood cells and all major organs of PrPKO mice relative to Wt controls. The iron deficient phenotype of PrPKO mice is reversed by expressing Wt PrP in the PrPKO background, demonstrating a functional role for PrP in iron uptake and transport. Since iron is required for essential metabolic processes and is also potentially toxic if mismanaged, these results suggest that loss of normal function of PrP due to aggregation to the PrPSc form induces imbalance of brain iron homeostasis, resulting in disease associated neurotoxicity. PMID:19568430

  7. TRP vanilloid 2 knock-out mice are susceptible to perinatal lethality but display normal thermal and mechanical nociception.

    PubMed

    Park, Una; Vastani, Nisha; Guan, Yun; Raja, Srinivasa N; Koltzenburg, Martin; Caterina, Michael J

    2011-08-10

    TRP vanilloid 2 (TRPV2) is a nonselective cation channel expressed prominently in medium- to large-diameter sensory neurons that can be activated by extreme heat (>52°C). These features suggest that TRPV2 might be a transducer of noxious heat in vivo. TRPV2 can also be activated by hypoosmolarity or cell stretch, suggesting potential roles in mechanotransduction. To address the physiological functions of TRPV2 in somatosensation, we generated TRPV2 knock-out mice and examined their behavioral and electrophysiological responses to heat and mechanical stimuli. TRPV2 knock-out mice showed reduced embryonic weight and perinatal viability. As adults, surviving knock-out mice also exhibited a slightly reduced body weight. TRPV2 knock-out mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation. Moreover, behavioral assays of TRPV1/TRPV2 double knock-out mice or of TRPV2 knock-out mice treated with resiniferatoxin to desensitize TRPV1-expressing afferents revealed no thermosensory consequences of TRPV2 absence. In line with behavioral findings, electrophysiological recordings from skin afferents showed that C-fiber responses to heat and C- and Aδ-fiber responses to noxious mechanical stimuli were unimpaired in the absence of TRPV2. The prevalence of thermosensitive Aδ-fibers was too low to permit comparison between genotypes. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse.

  8. Glutaminyl Cyclase Knock-out Mice Exhibit Slight Hypothyroidism but No Hypogonadism

    PubMed Central

    Schilling, Stephan; Kohlmann, Stephanie; Bäuscher, Christoph; Sedlmeier, Reinhard; Koch, Birgit; Eichentopf, Rico; Becker, Andreas; Cynis, Holger; Hoffmann, Torsten; Berg, Sabine; Freyse, Ernst-Joachim; von Hörsten, Stephan; Rossner, Steffen; Graubner, Sigrid; Demuth, Hans-Ulrich

    2011-01-01

    Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development. PMID:21330373

  9. Naloxone fails to produce conditioned place aversion in mu-opioid receptor knock-out mice.

    PubMed

    Skoubis, P D; Matthes, H W; Walwyn, W M; Kieffer, B L; Maidment, N T

    2001-01-01

    There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.

  10. Motivational effects of ethanol in DARPP-32 knock-out mice.

    PubMed

    Risinger, F O; Freeman, P A; Greengard, P; Fienberg, A A

    2001-01-01

    DARPP-32 (dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa) is an important component of dopaminergic function in brain areas thought to be important for drug and alcohol addiction. The present experiments characterized the acquisition of ethanol-induced conditioned taste aversion, ethanol-induced conditioned place preference, and ethanol self-administration in DARPP-32 knock-out (KO) mice compared to wild-type (WT) controls. For taste conditioning, KO and WT mice received access to 0.2 m NaCl solution followed immediately by intraperitoneal injection of 0-4 gm/kg ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. For place conditioning, KO and WT mice received eight pairings of a tactile stimulus with ethanol (2 gm/kg, i.p.), and a different stimulus with saline. Ethanol produced increases in locomotor activity during conditioning, with KO mice showing higher activity levels after ethanol compared to WT mice. WT mice, but not KO mice, acquired conditioned preference for the ethanol-paired stimulus. In the self-administration procedure, KO and WT mice were trained to lever press for access to 10% v/v ethanol. Subsequently, the mice had 23 hr/d access to food, ethanol, and water. Response patterns were determined using 0-30% v/v ethanol concentrations. WT mice displayed concentration-dependent responding for ethanol. Responding on the ethanol lever by KO mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Ethanol responding increased in both genotypes, although WT mice showed higher rates at all concentrations.

  11. Age-dependent deficits in fear learning in heterozygous BDNF knock-out mice.

    PubMed

    Endres, Thomas; Lessmann, Volkmar

    2012-11-15

    Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF knock-out mice (BDNF(+/-)). Since brain BDNF levels are known to decline with aging, we hypothesized that BDNF(+/-) mice might show reduced fear learning at older ages. Indeed, BDNF(+/-) animals revealed an age-dependent deficit in fear learning 3 mo after birth and beyond. Since there were no alterations between the two genotypes during the conditioning training and when testing short-term memory, this learning deficit most likely reflects a deficit in memory consolidation. Importantly, there were no differences in spontaneous motor behavior and baseline anxiety in BDNF(+/-) animals at any age tested. Following behavioral testing quantification of BDNF levels in the basolateral amygdala with a sensitive BDNF ELISA revealed a positive correlation between the levels of BDNF in the amygdala and the individual learning performance. However, the age-dependent decline in the efficiency of fear conditioning in BDNF(+/-) mice was not accompanied by reduced BDNF expression in the amygdala. Thus, while reduced BDNF levels in general correlate with less efficient fear learning, this lack of BDNF can be compensated in young but not in older animals, suggesting that the cellular mechanisms responsible for fear learning consolidation become BDNF-dependent 3 mo after birth.

  12. Reduced cortical BDNF expression and aberrant memory in Carf knock-out mice.

    PubMed

    McDowell, Kelli A; Hutchinson, Ashley N; Wong-Goodrich, Sarah J E; Presby, Matthew M; Su, Dan; Rodriguiz, Ramona M; Law, Krystal C; Williams, Christina L; Wetsel, William C; West, Anne E

    2010-06-02

    Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-response factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding brain-derived neurotrophic factor (Bdnf). We have now generated Carf knock-out (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex, whereas BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wild-type littermates. Finally, Carf KO mice show normal short-term (STM) and long-term memory (LTM) in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Together, these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory.

  13. Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice*

    PubMed Central

    Tomilov, Alexey A.; Bicocca, Vincent; Schoenfeld, Robert A.; Giorgio, Marco; Migliaccio, Enrica; Ramsey, Jon J.; Hagopian, Kevork; Pelicci, Pier Giuseppe; Cortopassi, Gino A.

    2010-01-01

    A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(−/−) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(−/−) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cδ, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity. PMID:19892704

  14. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy

    PubMed Central

    Chadi, Sead; Polyte, Jacqueline; Lefevre, Lucas; Castille, Johan; Ehanno, Aude; Laubier, Johann; Jaffrézic, Florence; Le Provost, Fabienne

    2016-01-01

    R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1’s partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function. PMID:27611670

  15. Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.

    PubMed

    Akil, Omar; Chang, Jolie; Hiel, Hakim; Kong, Jee-Hyun; Yi, Eunyoung; Glowatzki, Elisabeth; Lustig, Lawrence R

    2006-12-13

    After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.

  16. Mildly Increased Mechanical Nociceptive Sensitivity in REV-ERBα Knock-out Mice

    PubMed Central

    Lee, Jaehyun; Ko, Hyoung-Gon; Kim, Kyungjin

    2016-01-01

    Nociception is one of the most complex senses that is affected not only by external stimulation but also internal conditions. Previous studies have suggested that circadian rhythm is important in modulating nociception. REV-ERBα knock-out (KO) mice have disrupted circadian rhythm and altered mood-related phenotypes. In this study, we examined the role of REV-ERBα in inflammatory nociception. We found that the nociceptive sensitivity of KO mice was partially enhanced in mechanical nociception. However, this partial alteration was independent of the circadian rhythm. Taken together, deletion of REV-ERBα induced a mild change in mechanical nociceptive sensitivity but this alteration was not dependent on the circadian rhythm. PMID:28035185

  17. Schmallenberg virus infection of adult type I interferon receptor knock-out mice.

    PubMed

    Wernike, Kerstin; Breithaupt, Angele; Keller, Markus; Hoffmann, Bernd; Beer, Martin; Eschbaumer, Michael

    2012-01-01

    Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Europe in late 2011. It causes mild and transient disease in adult ruminants, but fetal infection can lead to abortion or severe malformations. There is considerable demand for SBV research, but in vivo studies in large animals are complicated by their long gestation periods and the cost of high containment housing. The goal of this study was to investigate whether type I interferon receptor knock-out (IFNAR(-/-)) mice are a suitable small animal model for SBV. Twenty IFNAR(-/-) mice were inoculated with SBV, four were kept as controls. After inoculation, all were observed and weighed daily; two mice per day were sacrificed and blood, brain, lungs, liver, spleen, and intestine were harvested. All but one inoculated mouse lost weight, and two mice died spontaneously at the end of the first week, while another two had to be euthanized. Real-time RT-PCR detected large amounts of SBV RNA in all dead or sick mice; the controls were healthy and PCR-negative. IFNAR(-/-) mice are susceptible to SBV infection and can develop fatal disease, making them a handy and versatile tool for SBV vaccine research.

  18. Effects of cinnarizine, a calcium antagonist that produces human parkinsonism, in parkin knock out mice.

    PubMed

    Serrano, A; Menéndez, J; Casarejos, M J; Solano, R M; Gallego, E; Sánchez, M; Mena, M A; García de Yebenes, J

    2005-08-01

    Cinnarizine, a calcium antagonist that produces parkinsonism in humans, induces behavioural changes such as alopecia, buco-lingual dyskinesia and reduction of motor activity in female parkin knock out (PK-KO) mice but not in wild-type (WT) controls. PK-KO mice have high striatal dopamine levels and increased dopamine metabolism in spite of low reduced tyrosine hydroxylase protein. Cinnarizine, which blocks dopamine receptors and increases dopamine release, further increased dopamine metabolism. PK-KO mice increased GSH levels as a compensatory mechanism against enhanced free radical production related to acceleration of dopamine turnover. Neuronal markers, such as beta-tubulin slightly increased in PK-KO and furthermore with cinnarizine. Astroglial markers were decreased in PK-KO mice, and this effect was potentiated by cinnarizine, suggesting abnormal glia in these animals. Microglia was hyperactivated in PK-KO midbrain, suggesting inflammation in these animals. Proapoptotic proteins were increased by cinnarizine and, to a lesser extent, in PK-KO mice. Our data indicate that mutation of parkin is a risk factor for drug-induced parkinsonism.

  19. Impaired sensorimotor gating in Fmr1 knock out and Fragile X premutation model mice.

    PubMed

    Renoux, A J; Sala-Hamrick, K J; Carducci, N M; Frazer, M; Halsey, K E; Sutton, M A; Dolan, D F; Murphy, G G; Todd, P K

    2014-07-01

    Fragile X syndrome (FXS) is a common inherited cause of intellectual disability that results from a CGG repeat expansion in the FMR1 gene. Large repeat expansions trigger both transcriptional and translational suppression of Fragile X protein (FMRP) production. Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is an allelic neurodegenerative disease caused by smaller "pre-mutation" CGG repeat expansions that enhance FMR1 transcription but lead to translational inefficiency and reduced FMRP expression in animal models. Sensorimotor gating as measured by pre-pulse inhibition (PPI) is altered in both FXS patients and Fmr1 knock out (KO) mice. Similarly, FXTAS patients have demonstrated PPI deficits. Recent work suggests there may be overlapping synaptic defects between Fmr1 KO and CGG knock-in premutation mouse models (CGG KI). We therefore sought to interrogate PPI in CGG KI mice. Using a quiet PPI protocol more akin to human testing conditions, we find that Fmr1 KO animals have significantly impaired PPI. Using this same protocol, we find CGG KI mice demonstrate an age-dependent impairment in PPI compared to wild type (WT) controls. This study describes a novel phenotype in CGG KI mice that can be used in future therapeutic development targeting premutation associated symptoms.

  20. Uptake and catabolism of modified LDL in scavenger-receptor class A type I/II knock-out mice.

    PubMed Central

    Van Berkel, T J; Van Velzen, A; Kruijt, J K; Suzuki, H; Kodama, T

    1998-01-01

    The liver is the major organ responsible for the uptake of modified low-density lipoprotein (LDL) from the blood circulation, with endothelial and Kupffer cells as major cellular uptake sites. Scavenger-receptors, which include various classes, are held responsible for this uptake. Mice deficient in scavenger-receptor class A types I and II were created and the fate of acetylated LDL (Ac-LDL) in vivo and its interaction with liver endothelial, Kupffer and peritoneal macrophages was characterized. Surprisingly, the decay in vivo (t12 < 2 min), tissue distribution and liver uptake (at 5 min it was 77.4 +/- 4.6% of the injected dose) of Ac-LDL in the knock-out mice were not significantly different from control mice (t12 < 2 min and liver uptake 79.1 +/- 4.6% of the injected dose). A separation of mice liver cells into parenchymal, endothelial and Kupffer cells 10 min after injection of Ac-LDL indicated that in both control and knock-out mice the liver endothelial cells were responsible for more than 70% of the liver uptake. Both in control and knock-out mice, preinjection of polyinosinic acid (poly I, 200 microg) completely blocked the liver uptake, indicating that both in control and knock-out mice the scavenger-receptors are sensitive to poly I. Preinjection of suboptimal poly I concentrations (20 and 50 microg) provided evidence that the serum decay and liver uptake of Ac-LDL is more readily inhibited in the knock-out mice as compared with the control mice, indicating less efficient removal of Ac-LDL in vivo in the knock-out mice under these conditions. Studies in vitro with isolated liver endothelial and Kupffer cells from knock-out mice indicate that the cell association of Ac-LDL during 2 h at 37 degrees C is 50 and 53% of the control, respectively, whereas the degradation reaches values of 58 and 63%. For peritoneal macrophages from knock-out mice the cell association of Ac-LDL was identical to the control mice whereas the Ac-LDL degradation in cells from the

  1. Knock-out of nexilin in mice leads to dilated cardiomyopathy and endomyocardial fibroelastosis.

    PubMed

    Aherrahrou, Zouhair; Schlossarek, Saskia; Stoelting, Stephanie; Klinger, Matthias; Geertz, Birgit; Weinberger, Florian; Kessler, Thorsten; Aherrahrou, Redouane; Moreth, Kristin; Bekeredjian, Raffi; Hrabě de Angelis, Martin; Just, Steffen; Rottbauer, Wolfgang; Eschenhagen, Thomas; Schunkert, Heribert; Carrier, Lucie; Erdmann, Jeanette

    2016-01-01

    Cardiomyopathy is one of the most common causes of chronic heart failure worldwide. Mutations in the gene encoding nexilin (NEXN) occur in patients with both hypertrophic and dilated cardiomyopathy (DCM); however, little is known about the pathophysiological mechanisms and relevance of NEXN to these disorders. Here, we evaluated the functional role of NEXN using a constitutive Nexn knock-out (KO) mouse model. Heterozygous (Het) mice were inter-crossed to produce wild-type (WT), Het, and homozygous KO mice. At birth, 32, 46, and 22 % of the mice were WT, Het, and KO, respectively, which is close to the expected Mendelian ratio. After postnatal day 6, the survival of the Nexn KO mice decreased dramatically and all of the animals died by day 8. Phenotypic characterizations of the WT and KO mice were performed at postnatal days 1, 2, 4, and 6. At birth, the relative heart weights of the WT and KO mice were similar; however, at day 4, the relative heart weight of the KO group was 2.3-fold higher than of the WT group. In addition, the KO mice developed rapidly progressive cardiomyopathy with left ventricular dilation and wall thinning and decreased cardiac function. At day 6, the KO mice developed a fulminant DCM phenotype characterized by dilated ventricular chambers and systolic dysfunction. At this stage, collagen deposits and some elastin deposits were observed within the left ventricle cavity, which resembles the features of endomyocardial fibroelastosis (EFE). Overall, these results further emphasize the role of NEXN in DCM and suggest a novel role in EFE.

  2. Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Iijima, Mikio; Li, Meng Xian; Kobayashi, Keiko; Horiuchi, Masahisa; Ushikai, Miharu; Okumura, Fumihiko; Meng, Xiao Jian; Inoue, Ituro; Tajima, Atsushi; Moriyama, Mitsuaki; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Tsui, Lap-Chee; Tsuji, Mihoko; Okano, Akira; Kobayashi, Tsuyoshi

    2007-08-24

    Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

  3. Oral rapamycin inhibits growth of atherosclerotic plaque in apoE knock-out mice

    SciTech Connect

    Waksman, Ron; Pakala, Rajbabu; Burnett, Mary S.; Gulick, Cindy P.; Leborgne, Laurent; Fournadjiev, Jana; Wolfram, Roswitha; Hellinga, David

    2003-03-01

    Introduction: Inflammatory and immunological responses of vascular cells are known to play significant roles in atherosclerotic plaque development. Rapamycin with antiinflammatory, immunosuppressive and antiproliferative properties has been shown to reduce neointima formation when coated on stents. This study is designed to test the potential of oral rapamycin to inhibit atherosclerotic plaque development. Methods: Eight-week-old apoE knock-out mice were fed with 0.25% cholesterol supplemented diet (control diet), control diet containing 50 {mu}g/kg rapamycin (low-dose rapamycin) or 100 {mu}g/kg rapamycin (high-dose rapamycin) for 4 or 8 weeks. Subsets of mice from each group (n=10) were weighed and euthanized. Whole blood rapamycin levels were determined using HPLC-MS/MS, and histological analyses of atherosclerotic lesions in the aortic root were performed. Results: Mice fed with high-dose rapamycin did not gain weight (18.5{+-}1.5 vs. 20.6{+-}0.9 g, P=.01). Blood levels of rapamycin 117{+-}7 pg/ml were detected in the blood of mice fed with high-dose rapamycin for 8 weeks. The plaque area in mice fed with high dose oral rapamycin is significantly less as compared to control (0.168{+-}0.008 vs. 0.326{+-}0.013 mm{sup 2}, P=.001 at 4 weeks; 0.234{+-}0.013 vs. 0.447{+-}0.011 mm{sup 2}, P=.001 at 8 weeks). Lumen area was inversely proportional to the plaque area. Conclusions: The results indicate that oral rapamycin is effective in attenuating the progression of atherosclerotic plaque in the mice.

  4. Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development.

    PubMed

    Schilling, Stephan; Kohlmann, Stephanie; Bäuscher, Christoph; Sedlmeier, Reinhard; Koch, Birgit; Eichentopf, Rico; Becker, Andreas; Cynis, Holger; Hoffmann, Torsten; Berg, Sabine; Freyse, Ernst-Joachim; von Hörsten, Stephan; Rossner, Steffen; Graubner, Sigrid; Demuth, Hans-Ulrich

    2011-04-22

    Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.

  5. Systemic and Cerebral Iron Homeostasis in Ferritin Knock-Out Mice

    PubMed Central

    Li, Wei; Garringer, Holly J.; Goodwin, Charles B.; Richine, Briana; Acton, Anthony; VanDuyn, Natalia; Muhoberac, Barry B.; Irimia-Dominguez, Jose; Chan, Rebecca J.; Peacock, Munro; Nass, Richard; Ghetti, Bernardino; Vidal, Ruben

    2015-01-01

    Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect. PMID:25629408

  6. Subregion-specific p300 conditional knock-out mice exhibit long-term memory impairments.

    PubMed

    Oliveira, Ana M M; Estévez, Marcel A; Hawk, Joshua D; Grimes, Shannon; Brindle, Paul K; Abel, Ted

    2011-01-01

    Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting proteins including transcription factors known to play a role in long-term memory formation. Thus, CBP and p300 constitute likely candidates for transcriptional coactivators in memory formation. In this study, we took a loss-of-function approach to evaluate the role of p300 in long-term memory formation. We used conditional knock-out mice in which the deletion of p300 is restricted to the postnatal phase and to subregions of the forebrain. We found that p300 is required for the formation of long-term recognition memory and long-term contextual fear memory in the CA1 area of the hippocampus and cortical areas.

  7. Purinergic neuromuscular transmission is absent in the colon of P2Y1 knocked out mice

    PubMed Central

    Gallego, Diana; Gil, Víctor; Martínez-Cutillas, Míriam; Mañé, Noemí; Martín, Maria Teresa; Jiménez, Marcel

    2012-01-01

    Purinergic and nitrergic co-transmission is the dominant mechanism responsible for neural-mediated smooth muscle relaxation in the gastrointestinal tract. The aim of the present paper was to test whether or not P2Y1 receptors are involved in purinergic neurotransmission using P2Y1−/− knock-out mice. Tension and microelectrode recordings were performed on colonic strips. In wild type (WT) animals, electrical field stimulation (EFS) caused an inhibitory junction potential (IJP) that consisted of a fast IJP (MRS2500 sensitive, 1 μm) followed by a sustained IJP (Nω-nitro-l-arginine (l-NNA) sensitive, 1 mm). The fast component of the IJP was absent in P2Y1−/− mice whereas the sustained IJP (l-NNA sensitive) was recorded. In WT animals, EFS-induced inhibition of spontaneous motility was blocked by the consecutive addition of l-NNA and MRS2500. In P2Y1−/− mice, EFS responses were completely blocked by l-NNA. In WT and P2Y1−/− animals, l-NNA induced a smooth muscle depolarization but ‘spontaneous’ IJP (MRS2500 sensitive) could be recorded in WT but not in P2Y1−/− animals. Finally, in WT animals, 1 μm MRS2365 caused a smooth muscle hyperpolarization that was blocked by 1 μm MRS2500. In contrast, 1 μm MRS2365 did not modify smooth muscle resting membrane potential in P2Y1−/− mice. β-Nicotinamide adenine dinucleotide (β-NAD, 1 mm) partially mimicked the effect of MRS2365. We conclude that P2Y1 receptors mediate purinergic neurotransmission in the gastrointestinal tract and β-NAD partially fulfils the criteria to participate in rodent purinergic neurotransmission. The P2Y1−/− mouse is a useful animal model to study the selective loss of purinergic neurotransmission. PMID:22371472

  8. Prolonged Starvation Causes Up-Regulation of AQP1 in Adipose Tissue Capillaries of AQP7 Knock-Out Mice

    PubMed Central

    Skowronski, Mariusz T.; Skowronska, Agnieszka; Rojek, Aleksandra; Oklinski, Michal K.; Nielsen, Søren

    2016-01-01

    Aquaporins (AQPs) are membrane proteins involved in the regulation of cellular transport and the balance of water and glycerol and cell volume in the white adipose tissue (WAT). In our previous study, we found the co-expression of the AQP1 water channel and AQP7 in the mouse WAT. In our present study, we aimed to find out whether prolonged starvation influences the AQP1 expression of AQP7 knock-out mice (AQP7 KO) in the WAT. To resolve this hypothesis, immunoperoxidase, immunoblot and immunogold microscopy were used. AQP1 expression was found with the use of immunohistochemistry and was confirmed by immunogold microscopy in the vessels of mouse WAT of all studied groups. Semi-quantitative immunoblot and quantitative immunogold microscopy showed a significant increase (by 2.5- to 3-fold) in the abundance of AQP1 protein expression in WAT in the 72 h starved AQP7 KO mice as compared to AQP7+/+ (p < 0.05) and AQP7−/− (p < 0.01) controls, respectively. In conclusion, the AQP1 water channel located in the vessels of WAT is up-regulated in response to prolonged starvation in the WAT of AQP7 KO mice. The present data suggest that an interaction of different AQP isoforms is required for maintaining proper water homeostasis within the mice WAT. PMID:27455244

  9. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

    PubMed

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

    2013-01-01

    Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

  10. Mouse Nuclear Myosin I Knock-Out Shows Interchangeability and Redundancy of Myosin Isoforms in the Cell Nucleus

    PubMed Central

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

    2013-01-01

    Background Nuclear myosin I (NM1) is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes. PMID:23593477

  11. Smad3 knock-out mice as a useful model to study intestinal fibrogenesis

    PubMed Central

    Zanninelli, Giuliana; Vetuschi, Antonella; Sferra, Roberta; D’Angelo, Angela; Fratticci, Amato; Continenza, Maria Adelaide; Chiaramonte, Maria; Gaudio, Eugenio; Caprilli, Renzo; Latella, Giovanni

    2006-01-01

    AIM: To evaluate the possible differences in morphology and immunohistochemical expression of CD3, transforming growth factor β1(TGF-β1), Smad7, α-smooth muscle actin (α-Sma), and collagen types I-VII of small and large intestine in Smad3 null and wild-type mice. METHODS: Ten null and ten wild-type adult mice were sacrificed at 4 mo of age and the organs (esophagus, small and large bowel, ureters) were collected for histology(hematoxylin and eosin, Masson thrichrome, silver staining), morphometry and immunohistochemistry analysis. TGF-β1 levels of intestinal tissue homogenates were assessed by ELISA. RESULTS: No macroscopic intestinal lesions were detected both in null and wild-type mice. Histological and morphometric evaluation revealed a significant reduction in muscle layer thickness of small and large intestine in null mice as compared to wild-type mice. Immunohistochemistry evaluation showed a significant increase of CD3+T cell, TGF-β1 and Smad7 staining in the small and large intestine mucosa of Smad3 null mice as compared to wild-type mice. α-Sma and collagen I-VII staining of small and large intestine did not differ between the two groups of mice. TGF-β1 levels of colonic tissue homogenates were significantly higher in null mice than in wild-type mice. In preliminary experiments a significant reduction of TNBS-induced intestinal fibrosis was observed in null mice as compared to wild-type mice. CONCLUSION: Smad3 null mice are a useful model to investigate the in vivo role of the TGF-β/Smad signalling pathway in intestinal inflammation and fibrosis. PMID:16534873

  12. Behavioral and cognitive data in mice with different tryptophan-metabolizing enzymes knocked out.

    PubMed

    Too, Lay Khoon; Li, Kong M; Suarna, Cacang; Maghzal, Ghassan J; Stocker, Roland; McGregor, Iain S; Hunt, Nicholas H

    2016-12-01

    This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study "Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function" (Too LK, Li KM, Suarna C, Maghzal GJ, Stocker R, McGregor IS, et al., 2016) [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access - the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.

  13. Three-dimensional reconstruction of efferent ducts in wild-type and Lgr4 knock-out mice.

    PubMed

    Lambot, Marie-Alexandra H; Mendive, Fernando; Laurent, Patrick; Van Schoore, Gregory; Noël, Jean-Christophe; Vanderhaeghen, Pierre; Vassart, Gilbert

    2009-04-01

    We have recently shown that Lgr4 knock-out (LGR4KO) male mice are infertile due to a developmental defect of the reproductive tract. Spermatozoa do not reach the epididymis and accumulate at the rete testis and efferent ducts (ED). We have proposed that in LGR4KO, ED might fail to connect resulting in blind-ended tubes that preclude the normal transit of sperm cells. To explore this possibility, we reconstructed the three-dimensional (3D) structure of the organ from serial microphotographs. The resulting model allowed to individualize and follow each ED from the testis up to the epididymis, and to display the spatial distribution of their content. The transit of spermatozoa is indeed blocked in LGR4KO mice but, contrary to the expectation, the ducts connect normally to each other, forming a single tube that flows into the epididymis, as in the wild-type animals. In the KO however, transit of the sperm is abruptly blocked at the same level syncytial-like aggregates appear in the luminal space. The model also allowed calculating, for the first time, morphometric parameters of the mouse ED, such as total volume, surface, radius, and length. These data unambiguously showed that ED in the mutant mouse are dramatically shortened and less convoluted than in the wild-type animal, providing an explanation to the phenotype observed in LGR4KO. Combined with in situ immunodetection or RNA in situ hybridization, 3D reconstruction of serial histological sections will provide an efficient mean to study expression profiles in organs which do not lend themselves to whole-mount studies.

  14. C6 knock-out Mice Are Protected from Thrombophilia Mediated by Antiphospholipid Antibodies

    PubMed Central

    Laura, Carrera-Marín Ana; Zurina, Romay-Penabad; Elizabeth, Papalardo; Elba, Reyes-Maldonado; Ethel, Garcia-Latorre; Gracie, Vargas; Tuya, Shilagard; Silvia, Pierangeli

    2013-01-01

    Background Complement activation plays a role in pathogenesis of the Antiphospholipid Syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6-/-) mice. Methods C6-/- or the wild-type (C3H/HeJ) C6+/+ mice were injected twice with IgG-APS (n=2) or IgM-APS (n=1) isolated from APS patients or with the corresponding control Igs (IgG-NHS or IgM-NHS). Then, the size of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. Results Thrombus sizes were significantly larger in C6+/+ treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6+/+ mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6-/- mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p<0.001), compared to their C6+/+ counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6-/- mice treated with IgG-APS were diminished when compared to C6+/+ treated with the same immunoglobulins. All mice injected with IgG-APS and IgM-APS had medium-high titers of aCL and aβ2GPI antibodies. Conclusions These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations. PMID:22933620

  15. Autistic-Like Traits and Cerebellar Dysfunction in Purkinje Cell PTEN Knock-Out Mice.

    PubMed

    Cupolillo, Dario; Hoxha, Eriola; Faralli, Alessio; De Luca, Annarita; Rossi, Ferdinando; Tempia, Filippo; Carulli, Daniela

    2016-05-01

    Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits.

  16. Knock out of the NADPH oxidase Nox4 has no impact on life span in mice.

    PubMed

    Rezende, Flavia; Schürmann, Christoph; Schütz, Susanne; Harenkamp, Sabine; Herrmann, Eva; Seimetz, Michael; Weißmann, Norbert; Schröder, Katrin

    2017-04-01

    The free radical theory of aging suggests reactive oxygen species as a main reason for accumulation of damage events eventually leading to aging. Nox4, a member of the family of NADPH oxidases constitutively produces ROS and therefore has the potential to be a main driver of aging. Herein we analyzed the life span of Nox4 deficient mice and found no difference when compared to their wildtype littermates. Accordingly neither Tert expression nor telomere length was different in cells isolated from those animals. In fact, Nox4 mRNA expression in lungs of wildtype mice dropped with age. We conclude that Nox4 has no influence on lifespan of healthy mice.

  17. Generation and Behavioral Characterization of β-catenin Forebrain-Specific Conditional Knock-Out Mice

    PubMed Central

    Gould, Todd D.; O'Donnell, Kelley C.; Picchini, Alyssa M.; Dow, Eliot R.; Chen, Guang; Manji, Husseini K.

    2009-01-01

    The canonical Wnt pathway and β-catenin have been implicated in the pathophysiology of mood disorders. We generated forebrain-specific CRE-mediated conditional β-catenin knockout mice to begin exploring the behavioral implications of decreased Wnt pathway signaling in the central nervous system. In situ hybridization revealed a progressive knockout of β-catenin that began between 2 and 4 weeks of age, and by 12 weeks resulted in considerably decreased β-catenin expression in regions of the forebrain, including the frontal cortex, hippocampus, and striatum. A significant decrease in protein levels of β-catenin in these brain regions was observed by western blot. Behavioral characterization of these mice in several tests (including the forced swim test, tail suspension test (TST), learned helplessness, response and sensitization to stimulants, and light/dark box among other tests) revealed relatively circumscribed alterations. In the TST, knockout mice spent significantly less time struggling (a depression-like phenotype). However, knockout mice did not differ from their wild-type littermates in the other behavioral tests of mood-related or anxiety-related behaviors. These results suggest that a considerable β-catenin reserve exists, and that a 50-70% β-catenin reduction in circumscribed brain regions is only capable of inducing subtle behavioral changes. Alternatively, regulating β-catenin may modulate drug effects rather than being a model of mood disorder pathophysiology per se. PMID:18299155

  18. Analysis of knock-out mice to determine the role of HPC-1/syntaxin 1A in expressing synaptic plasticity.

    PubMed

    Fujiwara, Tomonori; Mishima, Tatsuya; Kofuji, Takefumi; Chiba, Tomoki; Tanaka, Keiji; Yamamoto, Akitsugu; Akagawa, Kimio

    2006-05-24

    The protein HPC-1/syntaxin 1A is abundantly expressed in neurons and localized in the neuronal plasma membrane. It forms a complex with SNAP-25 (25 kDa synaptosomal-associated protein) and VAMP-2 (vesicle-associated membrane protein)/synaptobrevin called SNARE (a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex, which is considered essential for synaptic vesicle exocytosis; thus, HPC-1/syntaxin 1A is considered crucial for synaptic transmission. To examine the physiological function of HPC-1/syntaxin 1A in vivo, we produced knock-out (KO) mice by targeted gene disruption. Although HPC-1/syntaxin 1A expression was completely depleted without any effect on the expression of other SNARE proteins, the KO mice were viable. They grew normally, were fertile, and displayed no difference in appearance compared with control littermate. In cultured hippocampal neurons derived from the KO mice, the basic synaptic transmission in vitro was normal. However, the mutant mice had impaired long-term potentiation in the hippocampal slice. Also, although KO mice exhibited normal spatial memory in the hidden platform test, consolidation of conditioned fear memory was impaired. Interestingly, the KO mice had impaired conditioned fear memory extinction. These observations suggest that HPC-1/syntaxin 1A may be closely related to synaptic plasticity.

  19. Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

    SciTech Connect

    Malur, Anagha; Huizar, Isham; Wells, Greg; Barna, Barbara P.; Malur, Achut G.; Thomassen, Mary Jane

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Lentivirus-ABCG1 reduces lipid accumulation in lungs of GM-CSF knock-out mice. Black-Right-Pointing-Pointer Up-regulation of ABCG1 improves lung function. Black-Right-Pointing-Pointer Upregulation of ABCG1 improves surfactant metabolism. -- Abstract: We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) and the PPAR{gamma}-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPAR{gamma}. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPAR{gamma} plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p = 0.0005) increase in ABCG1 expression with no change of PPAR{gamma} or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by

  20. Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice.

    PubMed

    Irimia, Jose M; Meyer, Catalina M; Peper, Caron L; Zhai, Lanmin; Bock, Cheryl B; Previs, Stephen F; McGuinness, Owen P; DePaoli-Roach, Anna; Roach, Peter J

    2010-04-23

    Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

  1. Impaired Dendritic Development and Memory in Sorbs2 Knock-Out Mice.

    PubMed

    Zhang, Qiangge; Gao, Xian; Li, Chenchen; Feliciano, Catia; Wang, Dongqing; Zhou, Dingxi; Mei, Yuan; Monteiro, Patricia; Anand, Michelle; Itohara, Shigeyoshi; Dong, Xiaowei; Fu, Zhanyan; Feng, Guoping

    2016-02-17

    Intellectual disability is a common neurodevelopmental disorder characterized by impaired intellectual and adaptive functioning. Both environmental insults and genetic defects contribute to the etiology of intellectual disability. Copy number variations of SORBS2 have been linked to intellectual disability. However, the neurobiological function of SORBS2 in the brain is unknown. The SORBS2 gene encodes ArgBP2 (Arg/c-Abl kinase binding protein 2) protein in non-neuronal tissues and is alternatively spliced in the brain to encode nArgBP2 protein. We found nArgBP2 colocalized with F-actin at dendritic spines and growth cones in cultured hippocampal neurons. In the mouse brain, nArgBP2 was highly expressed in the cortex, amygdala, and hippocampus, and enriched in the outer one-third of the molecular layer in dentate gyrus. Genetic deletion of Sorbs2 in mice led to reduced dendritic complexity and decreased frequency of AMPAR-miniature spontaneous EPSCs in dentate gyrus granule cells. Behavioral characterization revealed that Sorbs2 deletion led to a reduced acoustic startle response, and defective long-term object recognition memory and contextual fear memory. Together, our findings demonstrate, for the first time, an important role for nArgBP2 in neuronal dendritic development and excitatory synaptic transmission, which may thus inform exploration of neurobiological basis of SORBS2 deficiency in intellectual disability. Copy number variations of the SORBS2 gene are linked to intellectual disability, but the neurobiological mechanisms are unknown. We found that nArgBP2, the only neuronal isoform encoded by SORBS2, colocalizes with F-actin at neuronal dendritic growth cones and spines. nArgBP2 is highly expressed in the cortex, amygdala, and dentate gyrus in the mouse brain. Genetic deletion of Sorbs2 in mice leads to impaired dendritic complexity and reduced excitatory synaptic transmission in dentate gyrus granule cells, accompanied by behavioral deficits in acoustic

  2. Impaired Dendritic Development and Memory in Sorbs2 Knock-Out Mice

    PubMed Central

    Zhang, Qiangge; Gao, Xian; Li, Chenchen; Feliciano, Catia; Wang, Dongqing; Zhou, Dingxi; Mei, Yuan; Monteiro, Patricia; Anand, Michelle; Itohara, Shigeyoshi; Dong, Xiaowei; Fu, Zhanyan

    2016-01-01

    Intellectual disability is a common neurodevelopmental disorder characterized by impaired intellectual and adaptive functioning. Both environmental insults and genetic defects contribute to the etiology of intellectual disability. Copy number variations of SORBS2 have been linked to intellectual disability. However, the neurobiological function of SORBS2 in the brain is unknown. The SORBS2 gene encodes ArgBP2 (Arg/c-Abl kinase binding protein 2) protein in non-neuronal tissues and is alternatively spliced in the brain to encode nArgBP2 protein. We found nArgBP2 colocalized with F-actin at dendritic spines and growth cones in cultured hippocampal neurons. In the mouse brain, nArgBP2 was highly expressed in the cortex, amygdala, and hippocampus, and enriched in the outer one-third of the molecular layer in dentate gyrus. Genetic deletion of Sorbs2 in mice led to reduced dendritic complexity and decreased frequency of AMPAR-miniature spontaneous EPSCs in dentate gyrus granule cells. Behavioral characterization revealed that Sorbs2 deletion led to a reduced acoustic startle response, and defective long-term object recognition memory and contextual fear memory. Together, our findings demonstrate, for the first time, an important role for nArgBP2 in neuronal dendritic development and excitatory synaptic transmission, which may thus inform exploration of neurobiological basis of SORBS2 deficiency in intellectual disability. SIGNIFICANCE STATEMENT Copy number variations of the SORBS2 gene are linked to intellectual disability, but the neurobiological mechanisms are unknown. We found that nArgBP2, the only neuronal isoform encoded by SORBS2, colocalizes with F-actin at neuronal dendritic growth cones and spines. nArgBP2 is highly expressed in the cortex, amygdala, and dentate gyrus in the mouse brain. Genetic deletion of Sorbs2 in mice leads to impaired dendritic complexity and reduced excitatory synaptic transmission in dentate gyrus granule cells, accompanied by

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

    PubMed

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

    2016-07-06

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

  4. Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis*♦

    PubMed Central

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

    2014-01-01

    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

  5. Behavioral Phenotype of Fmr1 Knock-Out Mice during Active Phase in an Altered Light/Dark Cycle.

    PubMed

    Saré, R Michelle; Levine, Merlin; Smith, Carolyn Beebe

    2016-01-01

    Fragile X syndrome (FXS) is the most commonly inherited form of intellectual disability and is a disorder that is also highly associated with autism. FXS occurs as a result of an expanded CGG repeat sequence leading to transcriptional silencing. In an animal model of FXS in which Fmr1 is knocked out (Fmr1 KO), many physical, physiological, and behavioral characteristics of the human disease are recapitulated. Prior characterization of the mouse model was conducted during the day, the inactive phase of the circadian cycle. Circadian rhythms are an important contributor to behavior and may play a role in the study of disease phenotype. Moreover, changes in the parameters of circadian rhythm are known to occur in FXS animal models. We conducted an investigation of key behavioral phenotypes in Fmr1 KO mice during their active phase. We report that phase did not alter the Fmr1 KO phenotype in open field activity, anxiety, and learning and memory. There was a slight effect of phase on social behavior as measured by time in chamber, but not by time spent sniffing. Our data strengthen the existing data characterizing the phenotype of Fmr1 KO mice, indicating that it is independent of circadian phase.

  6. [Role of elastin in the development of vascular function. Knock-out study of the elastin gene in mice].

    PubMed

    Faury, G

    2001-01-01

    The elastic fibres endow extensible tissues with resiliency, such as in blood vessels, heart, skin and lung. Elastic fibres are made of microfibrils, and mainly elastin (90%) which provides the fibre with elasticity. Beside the biomechanical role of elastin, a close correlation between elastin and elastic fibre network disorganisation and vascular smooth muscle cell (VSMC) growth disregulation has been known for several years through the description and study of several human or animal polyfeatured or obstructive vascular diseases, such as supravalvular aortic stenosis (SVAS) and Williams syndrome (WS), both related to heterozygous mutations or deletion in the elastin gene. The study of mice knock-out for the elastin gene (homozygous or heterozygous) leads to think that elastin should now be seen as an important elastic component providing extensible tissues with resiliency, as well as a major developmental regulator of VSMC life cycle and smooth muscle tissue organisation. Further developments in the area of preventive therapy of SVAS, WS or other inherited muscular disorders are likely to arise from these results.

  7. Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice.

    PubMed

    Guidotti, J E; Mignon, A; Haase, G; Caillaud, C; McDonell, N; Kahn, A; Poenaru, L

    1999-05-01

    The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.

  8. Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

    PubMed

    Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

    2016-10-01

    This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1(+/+) control group (group A, n=6); SIRT1(+/+) osteoarthritis group (group B, n=6); SIRT1(-/-) control group (group C, n=6); SIRT1(-/-) osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1(-/-) osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1(+/+) osteoarthritis group and SIRT1(-/-) control group, SIRT1 protein expression was not obviously changed in the SIRT1(-/-) osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

  9. 2-Methyl-6-(phenylethynyl) pyridine (MPEP) reverses maze learning and PSD-95 deficits in Fmr1 knock-out mice

    PubMed Central

    Gandhi, Réno M.; Kogan, Cary S.; Messier, Claude

    2014-01-01

    Fragile X Syndrome (FXS) is caused by the lack of expression of the fragile X mental retardation protein (FMRP), which results in intellectual disability and other debilitating symptoms including impairment of visual-spatial functioning. FXS is the only single-gene disorder that is highly co-morbid with autism spectrum disorder and can therefore provide insight into its pathophysiology. Lack of FMRP results in altered group I metabotropic glutamate receptor (mGluR) signaling, which is a target for putative treatments. The Hebb-Williams (H-W) mazes are a set of increasingly complex spatial navigation problems that depend on intact hippocampal and thus mGluR-5 functioning. In the present investigation, we examined whether an antagonist of mGluR-5 would reverse previously described behavioral deficits in fragile X mental retardation 1 knock-out (Fmr1 KO) mice. Mice were trained on a subset of the H-W mazes and then treated with either 20 mg/kg of an mGluR-5 antagonist, 2-Methyl-6-(phenylethynyl) pyridine (MPEP; n = 11) or an equivalent dose of saline (n = 11) prior to running test mazes. Latency and errors were dependent variables recorded during the test phase. Immediately after completing each test, marble-burying behavior was assessed, which confirmed that the drug treatment was pharmacologically active during maze learning. Although latency was not statistically different between the groups, MPEP treated Fmr1 KO mice made significantly fewer errors on mazes deemed more difficult suggesting a reversal of the behavioral deficit. MPEP treated mice were also less perseverative and impulsive when navigating mazes. Furthermore, MPEP treatment reversed post-synaptic density-95 (PSD-95) protein deficits in Fmr1 KO treated mice, whereas levels of a control protein (β-tubulin) remained unchanged. These data further validate MPEP as a potentially beneficial treatment for FXS. Our findings also suggest that adapted H-W mazes may be a useful tool to document alterations in

  10. Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice*

    PubMed Central

    Schwindinger, William F.; Mirshahi, Uyenlinh L.; Baylor, Kelly A.; Sheridan, Kathleen M.; Stauffer, Anna M.; Usefof, Stephanie; Stecker, Mark M.; Mirshahi, Tooraj; Robishaw, Janet D.

    2012-01-01

    The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ3 and γ7 subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3−/−Gng7−/− double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ3 subunit is a component of a Gi/o protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ7 subunit is a component of a Golf protein that is responsible for A2A adenosine or D1 dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process. PMID:22207761

  11. T cell responses in mammalian diaphanous-related formin mDia1 knock-out mice.

    PubMed

    Eisenmann, Kathryn M; West, Richard A; Hildebrand, Dagmar; Kitchen, Susan M; Peng, Jun; Sigler, Robert; Zhang, Jinyi; Siminovitch, Katherine A; Alberts, Arthur S

    2007-08-24

    Activated T cells rapidly assemble filamentous (F-) actin networks in response to ligation of the T cell receptor or upon interaction with adhesive stimuli in order to facilitate cell migration and the formation of the immune synapse. Branched filament assembly is crucial for this process and is dependent upon activation of the Arp2/3 complex by the actin nucleation-promoting factor Wiskott-Aldrich Syndrome protein (WASp). Genetic disruption of the WAS gene has been linked to hematopoietic malignancies and various cytopenias. Although the contributions of WASp and Arp2/3 to T cell responses are fairly well characterized, the role of the mammalian Diaphanous (mDia)-related formins, which both nucleate and processively elongate non-branched F-actin, has not been demonstrated. Here, we report the effects on T cell development and function following the knock out of the murine Drf1 gene encoding the canonical formin p140mDia1. Drf1(-/-) mice develop lymphopenia characterized by diminished T cell populations in lymphoid tissues. Consistent with a role for p140mDia1 in the regulation of the actin cytoskeleton, isolated Drf1(-/-) splenic T cells adhered poorly to extracellular matrix proteins and migration in response to chemotactic stimuli was completely abrogated. Both integrin and chemokine receptor expression was unaffected by Drf1(-/-) targeting. In response to proliferative stimuli, both thymic and splenic Drf1(-/-) T cells failed to proliferate; ERK1/2 activation was also diminished in activated Drf1(-/-) T cells. These data suggest a central role for p140mDia1 in vivo in dynamic cytoskeletal remodeling events driving normal T cell responses.

  12. Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment.

    PubMed

    Kim, Munkyung; Piaia, Alessandro; Shenoy, Neeta; Kagan, David; Gapp, Berangere; Kueng, Benjamin; Weber, Delphine; Dietrich, William; Ksiazek, Iwona

    2015-01-01

    Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.

  13. Impairment of catecholamine systems during induction of long-term potentiation at hippocampal CA1 synapses in HPC-1/syntaxin 1A knock-out mice.

    PubMed

    Mishima, Tatsuya; Fujiwara, Tomonori; Kofuji, Takefumi; Akagawa, Kimio

    2012-01-04

    The membrane protein HPC-1/syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/syntaxin 1A knock-out (STX1A(-/-)) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A(-/-) mice. Because HPC-1/syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A(-/-) mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/syntaxin 1A causes impairment of LTP induction.

  14. Hawthorn (Crataegus pinnatifida Bunge) leave flavonoids attenuate atherosclerosis development in apoE knock-out mice.

    PubMed

    Dong, Pengzhi; Pan, Lanlan; Zhang, Xiting; Zhang, Wenwen; Wang, Xue; Jiang, Meixiu; Chen, Yuanli; Duan, Yajun; Wu, Honghua; Xu, Yantong; Zhang, Peng; Zhu, Yan

    2017-02-23

    Hawthorn (Crataegus pinnatifida Bunge) leave have been used to treat cardiovascular diseases in China and Europe. Hawthorn leave flavonoids (HLF) are the main part of extraction. Whether hawthorn leave flavonoids could attenuate the development of atherosclerosis and the possible mechanism remain unknown. High-fat diet (HFD) mixed with HLF at concentrations of 5mg/kg and 20mg/kg were administered to apolipoprotein E (apoE) knock out mice. 16 weeks later, mouse serum was collected to determine the lipid profile while the mouse aorta dissected was prepared to measure the lesion area. Hepatic mRNA of genes involved in lipid metabolism were determined. Peritoneal macrophages were collected to study the impact of HLF on cholesterol efflux, formation of foam cell and the expression of ATP binding cassette transporter A1 (ABCA1). Besides, in vivo reverse cholesterol transport (RCT) was conducted. HLF attenuated the development of atherosclerosis that the mean atherosclerotic lesion area in en face aortas was reduced by 23.1% (P<0.05). In mice fed with 20mg/kg HLF, Total cholesterol (TC) level was decreased by 18.6% and very low density lipoprotein cholesterol plus low density lipoprotein cholesterol (VLDLc+LDLc) level were decreased by 23.1% whereas high density lipoprotein cholesterol (HDLc) and triglyceride (TG) levels were similar compared to that of the control group. Peroxisome proliferator activated receptor alpha (PPARα) mRNA was increased by 31.2% (P<0.05) and 60.9% (P<0.05) in mice fed with 5mg/kg and 20mg/kg HLF respectively. Sterol regulatory element binding protein-1c (SREBP-1c) was decreased by 59.3% in the group of 20mg/kg. Carnitine palmitoyl transferase 1 (CPT-1) mRNA level of 20mg/kg group was induced 66.7% (P<0.05). Superoxide dismutase 1 and 2 (SOD1 and SOD2) mRNA were induced 25.4% (P<0.05) and 71.4% (P<0.05) while induced by 36.3% (P<0.05) and 73.2% (P<0.05) in group of 20mg/kg. Glutathione peroxidase 3 (Gpx3) mRNA in the group of 20mg/kg was induced

  15. Exacerbation of spontaneous autoimmune nephritis following regulatory T cell depletion in B cell lymphoma 2-interacting mediator knock-out mice.

    PubMed

    Wang, Y M; Zhang, G Y; Wang, Y; Hu, M; Zhou, J J; Sawyer, A; Cao, Q; Wang, Y; Zheng, G; Lee, V W S; Harris, D C H; Alexander, S I

    2017-02-02

    Regulatory T cells (Tregs ) have been recognized as central mediators for maintaining peripheral tolerance and limiting autoimmune diseases. The loss of Tregs or their function has been associated with exacerbation of autoimmune disease. However, the temporary loss of Tregs in the chronic spontaneous disease model has not been investigated. In this study, we evaluated the role of Tregs in a novel chronic spontaneous glomerulonephritis model of B cell lymphoma 2-interacting mediator (Bim) knock-out mice by transient depleting Tregs . Bim is a pro-apoptotic member of the B cell lymphoma 2 (Bcl-2) family. Bim knock-out (Bim(-/-) ) mice fail to delete autoreactive T cells in thymus, leading to chronic spontaneous autoimmune kidney disease. We found that Treg depletion in Bim(-/-) mice exacerbated the kidney injury with increased proteinuria, impaired kidney function, weight loss and greater histological injury compared with wild-type mice. There was a significant increase in interstitial infiltrate of inflammatory cells, antibody deposition and tubular damage. Furthermore, the serum levels of cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17α, interferon (IFN)-γ and tumour necrosis factor (TNF)-α were increased significantly after Treg depletion in Bim(-/-) mice. This study demonstrates that transient depletion of Tregs leads to enhanced self-reactive T effector cell function followed by exacerbation of kidney disease in the chronic spontaneous kidney disease model of Bim-deficient mice.

  16. Type II Cochlear Ganglion Neurons Do Not Drive the Olivocochlear Reflex: Re-Examination of the Cochlear Phenotype in Peripherin Knock-Out Mice

    PubMed Central

    2016-01-01

    Abstract The cochlear nerve includes a small population of unmyelinated sensory fibers connecting outer hair cells to the brain. The functional role of these type II afferent neurons is controversial, because neurophysiological data are sparse. A recent study (Froud et al., 2015) reported that targeted deletion of peripherin, a type of neurofilament, eliminated type II afferents and inactivated efferent feedback to the outer hair cells, thereby suggesting that type II afferents were the sensory drive to this sound-evoked, negative-feedback reflex, the olivocochlear pathway. Here, we re-evaluated the cochlear phenotype in mice from the peripherin knock-out line and show that (1) type II afferent terminals are present in normal number and (2) olivocochlear suppression of cochlear responses is absent even when this efferent pathway is directly activated by shocks. We conclude that type II neurons are not the sensory drive for the efferent reflex and that peripherin deletion likely causes dysfunction of synaptic transmission between olivocochlear terminals and their peripheral targets. PMID:27570826

  17. Impaired Angiogenesis during Fracture Healing in GPCR Kinase 2 Interacting Protein-1 (GIT1) Knock Out Mice

    PubMed Central

    Menon, Prashanthi; Pang, Jinjiang; Ho, Hsin-Chiu; Shi, Shanshan; Xie, Chao; Smolock, Elaine; Yan, Chen; Zuscik, Michael J.; Berk, Bradford C.

    2014-01-01

    G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus. PMID:24586541

  18. BOLD Imaging in Awake Wild-Type and Mu-Opioid Receptor Knock-Out Mice Reveals On-Target Activation Maps in Response to Oxycodone

    PubMed Central

    Moore, Kelsey; Madularu, Dan; Iriah, Sade; Yee, Jason R.; Kulkarni, Praveen; Darcq, Emmanuel; Kieffer, Brigitte L.; Ferris, Craig F.

    2016-01-01

    Blood oxygen level dependent (BOLD) imaging in awake mice was used to identify differences in brain activity between wild-type, and Mu (μ) opioid receptor knock-outs (MuKO) in response to oxycodone (OXY). Using a segmented, annotated MRI mouse atlas and computational analysis, patterns of integrated positive and negative BOLD activity were identified across 122 brain areas. The pattern of positive BOLD showed enhanced activation across the brain in WT mice within 15 min of intraperitoneal administration of 2.5 mg of OXY. BOLD activation was detected in 72 regions out of 122, and was most prominent in areas of high μ opioid receptor density (thalamus, ventral tegmental area, substantia nigra, caudate putamen, basal amygdala, and hypothalamus), and focus on pain circuits indicated strong activation in major pain processing centers (central amygdala, solitary tract, parabrachial area, insular cortex, gigantocellularis area, ventral thalamus primary sensory cortex, and prelimbic cortex). Importantly, the OXY-induced positive BOLD was eliminated in MuKO mice in most regions, with few exceptions (some cerebellar nuclei, CA3 of the hippocampus, medial amygdala, and preoptic areas). This result indicates that most effects of OXY on positive BOLD are mediated by the μ opioid receptor (on-target effects). OXY also caused an increase in negative BOLD in WT mice in few regions (16 out of 122) and, unlike the positive BOLD response the negative BOLD was only partially eliminated in the MuKO mice (cerebellum), and in some case intensified (hippocampus). Negative BOLD analysis therefore shows activation and deactivation events in the absence of the μ receptor for some areas where receptor expression is normally extremely low or absent (off-target effects). Together, our approach permits establishing opioid-induced BOLD activation maps in awake mice. In addition, comparison of WT and MuKO mutant mice reveals both on-target and off-target activation events, and set an OXY brain

  19. BOLD Imaging in Awake Wild-Type and Mu-Opioid Receptor Knock-Out Mice Reveals On-Target Activation Maps in Response to Oxycodone.

    PubMed

    Moore, Kelsey; Madularu, Dan; Iriah, Sade; Yee, Jason R; Kulkarni, Praveen; Darcq, Emmanuel; Kieffer, Brigitte L; Ferris, Craig F

    2016-01-01

    Blood oxygen level dependent (BOLD) imaging in awake mice was used to identify differences in brain activity between wild-type, and Mu (μ) opioid receptor knock-outs (MuKO) in response to oxycodone (OXY). Using a segmented, annotated MRI mouse atlas and computational analysis, patterns of integrated positive and negative BOLD activity were identified across 122 brain areas. The pattern of positive BOLD showed enhanced activation across the brain in WT mice within 15 min of intraperitoneal administration of 2.5 mg of OXY. BOLD activation was detected in 72 regions out of 122, and was most prominent in areas of high μ opioid receptor density (thalamus, ventral tegmental area, substantia nigra, caudate putamen, basal amygdala, and hypothalamus), and focus on pain circuits indicated strong activation in major pain processing centers (central amygdala, solitary tract, parabrachial area, insular cortex, gigantocellularis area, ventral thalamus primary sensory cortex, and prelimbic cortex). Importantly, the OXY-induced positive BOLD was eliminated in MuKO mice in most regions, with few exceptions (some cerebellar nuclei, CA3 of the hippocampus, medial amygdala, and preoptic areas). This result indicates that most effects of OXY on positive BOLD are mediated by the μ opioid receptor (on-target effects). OXY also caused an increase in negative BOLD in WT mice in few regions (16 out of 122) and, unlike the positive BOLD response the negative BOLD was only partially eliminated in the MuKO mice (cerebellum), and in some case intensified (hippocampus). Negative BOLD analysis therefore shows activation and deactivation events in the absence of the μ receptor for some areas where receptor expression is normally extremely low or absent (off-target effects). Together, our approach permits establishing opioid-induced BOLD activation maps in awake mice. In addition, comparison of WT and MuKO mutant mice reveals both on-target and off-target activation events, and set an OXY brain

  20. Increased analgesic tolerance to acute morphine in fosB knock-out mice: a gender study.

    PubMed

    Solecki, Wojciech; Krowka, Tomasz; Kubik, Jakub; Kaczmarek, Leszek; Przewlocki, Ryszard

    2008-10-01

    The proteins of Fos family are a potential candidate to link molecular mechanisms of morphine action with behavioural effects such as morphine-induced reward, dependence and tolerance. We used both male and female mice lacking fosB gene to study its contribution to morphine effects. Morphine analgesia (tail-flick test) and hypothermia were studied using morphine at cumulative doses in morphine-naive and morphine-tolerant (tolerance induced by 24 h prior 100 mg/kg morphine administration) mice. FosB -/- mice, as compared to fosB +/+ mice, developed enhanced tolerance to morphine-induced analgesia. No effects of genotype or gender on tolerance to morphine-induced hypothermia were observed. These results suggest that fosB may be involved in the development of tolerance to morphine analgesia but not hypothermia. The gender study implicates that lack of FosB proteins in female fosB -/- mice enhanced morphine analgesic potency. In conclusion, we show that fosB gene is important to analgesia but not hypothermia phenotype indicating its role in morphine effects.

  1. Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

    PubMed Central

    Brehm, Martin; Guenther, Madlen; Linnartz-Gerlach, Bettina; Neumann, Harald; Witte, Otto W.; Frahm, Christiane

    2013-01-01

    Background Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. Methods and Results As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. Conclusions Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke. PMID:23301011

  2. Enhanced Histaminergic Neurotransmission and Sleep-Wake Alterations, a Study in Histamine H3-Receptor Knock-Out Mice

    PubMed Central

    Gondard, Elise; Anaclet, Christelle; Akaoka, Hidéo; Guo, Rui-Xian; Zhang, Mei; Buda, Colette; Franco, Patricia; Kotani, Hidehito; Lin, Jian-Sheng

    2013-01-01

    Long-term abolition of a brain arousal system impairs wakefulness (W), but little is known about the consequences of long-term enhancement. The brain histaminergic arousal system is under the negative control of H3-autoreceptors whose deletion results in permanent enhancement of histamine (HA) turnover. In order to determine the consequences of enhancement of the histaminergic system, we compared the cortical EEG and sleep-wake states of H3-receptor knockout (H3R−/−) and wild-type mouse littermates. We found that H3R−/−mice had rich phenotypes. On the one hand, they showed clear signs of enhanced HA neurotransmission and vigilance, i.e., a higher EEG θ power during spontaneous W and a greater extent of W or sleep restriction during behavioral tasks, including environmental change, locomotion, and motivation tests. On the other hand, during the baseline dark period, they displayed deficient W and signs of sleep deterioration, such as pronounced sleep fragmentation and reduced cortical slow activity during slow wave sleep (SWS), most likely due to a desensitization of postsynaptic histaminergic receptors as a result of constant HA release. Ciproxifan (H3-receptor inverse agonist) enhanced W in wild-type mice, but not in H3R−/−mice, indicating a functional deletion of H3-receptors, whereas triprolidine (postsynaptic H1-receptor antagonist) or α-fluoromethylhistidine (HA-synthesis inhibitor) caused a greater SWS increase in H3R−/− than in wild-type mice, consistent with enhanced HA neurotransmission. These sleep-wake characteristics and the obesity phenotypes previously reported in this animal model suggest that chronic enhancement of histaminergic neurotransmission eventually compromises the arousal system, leading to sleep-wake, behavioral, and metabolic disorders similar to those caused by voluntary sleep restriction in humans. PMID:23303066

  3. Enhanced histaminergic neurotransmission and sleep-wake alterations, a study in histamine H3-receptor knock-out mice.

    PubMed

    Gondard, Elise; Anaclet, Christelle; Akaoka, Hidéo; Guo, Rui-Xian; Zhang, Mei; Buda, Colette; Franco, Patricia; Kotani, Hidehito; Lin, Jian-Sheng

    2013-05-01

    Long-term abolition of a brain arousal system impairs wakefulness (W), but little is known about the consequences of long-term enhancement. The brain histaminergic arousal system is under the negative control of H3-autoreceptors whose deletion results in permanent enhancement of histamine (HA) turnover. In order to determine the consequences of enhancement of the histaminergic system, we compared the cortical EEG and sleep-wake states of H3-receptor knockout (H3R-/-) and wild-type mouse littermates. We found that H3R-/-mice had rich phenotypes. On the one hand, they showed clear signs of enhanced HA neurotransmission and vigilance, i.e., a higher EEG θ power during spontaneous W and a greater extent of W or sleep restriction during behavioral tasks, including environmental change, locomotion, and motivation tests. On the other hand, during the baseline dark period, they displayed deficient W and signs of sleep deterioration, such as pronounced sleep fragmentation and reduced cortical slow activity during slow wave sleep (SWS), most likely due to a desensitization of postsynaptic histaminergic receptors as a result of constant HA release. Ciproxifan (H3-receptor inverse agonist) enhanced W in wild-type mice, but not in H3R-/-mice, indicating a functional deletion of H3-receptors, whereas triprolidine (postsynaptic H1-receptor antagonist) or α-fluoromethylhistidine (HA-synthesis inhibitor) caused a greater SWS increase in H3R-/- than in wild-type mice, consistent with enhanced HA neurotransmission. These sleep-wake characteristics and the obesity phenotypes previously reported in this animal model suggest that chronic enhancement of histaminergic neurotransmission eventually compromises the arousal system, leading to sleep-wake, behavioral, and metabolic disorders similar to those caused by voluntary sleep restriction in humans.

  4. Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.

    PubMed

    Hu, Jan C-C; Hu, Yuanyuan; Smith, Charles E; McKee, Marc D; Wright, J Timothy; Yamakoshi, Yasuo; Papagerakis, Petros; Hunter, Graeme K; Feng, Jerry Q; Yamakoshi, Fumiko; Simmer, James P

    2008-04-18

    Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

  5. Effects of eggplant (Solanum melongena) on the atherogenesis and oxidative stress in LDL receptor knock out mice (LDLR(-/-)).

    PubMed

    Botelho, Françoise V; Enéas, Luciana R; Cesar, Giovana C; Bizzotto, Carolina S; Tavares, Erico; Oliveira, Fabrícia A; Gloria, M Beatriz A; Silvestre, Marialice P C; Arantes, Rosa M E; Alvarez-Leite, Jacqueline I

    2004-08-01

    Eggplant (Solanum melongena) has been used as hypocholesterolemic agent in many countries. However, few controlled studies were addressed to this subject and atherogenesis. We have evaluated the effect of eggplant on cholesterol metabolism and atherogenesis in LDLR(-/-) mice. Animals were fed on chow (n=17) or atherogenic (n=21) diet during 12 weeks receiving water (control) or eggplant extract. Liver, serum and fecal lipids, together with serum lipoproteins were measured. Oxidative stress was evaluated through conjugate diene formation and ox-LDL antibodies by enzyme immunoassay. Atherosclerotic lesions were measured in different sites of aorta. Total cholesterol and atherogenic lipoproteins did not decrease after eggplant intake. Animals receiving eggplant and chow diet showed increased anti-ox-LDL antibodies and a decreased lag phase of conjugated diene formation, indicating a higher oxidative stress than controls. No differences were seen in lesion area of aortic valve. Eggplant extract had high histamine and other amine levels that could enhance LDL oxidation and its endocytosis. Eggplant did not decrease plasma cholesterol nor prevent the development of atherosclerosis in LDLR(-/-) mice. Surprisingly, eggplant increased oxidative stress, representing a risk factor for atherosclerosis. These results did not support the use of eggplant extract as hypocholesterolemic agent.

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

    PubMed

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

    2013-01-01

    Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular nucleotides on ion transport in the thick ascending limb (TAL). In the medullary thick ascending limb (mTAL), basolateral P2X receptors markedly (~25%) inhibit NaCl absorption. Although this segment does express both apical and basolateral P2Y2 receptors, acute activation of the basolateral P2Y2 receptors had no apparent effect on transepithelial ion transport. Here we studied, if the absence of the P2Y2 receptor causes chronic alterations in mTAL NaCl absorption by comparing basal and AVP-stimulated transepithelial transport rates. We used perfused mouse mTALs to electrically measure NaCl absorption in juvenile (<35 days) and adult (>35 days) male mice. Using microelectrodes, we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and thus, transepithelial NaCl absorption (equivalent short circuit current, I'sc). We find that mTALs from adult wild type (WT) mice have significantly lower NaCl absorption rates when compared to mTALs from juvenile WT mice. This could be attributed to significantly higher Rtevalues in mTALs from adult WT mice. This pattern was not observed in mTALs from P2Y2 receptor knockout (KO) mice. In addition, adult P2Y2 receptor KO mTALs have significantly lower Vtevalues compared to the juvenile. No difference in absolute I'sc was observed when comparing mTALs from WT and KO mice. AVP stimulated the mTALs to similar increases of NaCl absorption irrespective of the absence of the P2Y2 receptor. No difference was observed in the medullary expression level of NKCC2 in between the genotypes. These data indicate that the lack of P2Y2 receptors does not cause substantial differences in resting and AVP-stimulated NaCl absorption

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

    PubMed Central

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

    2013-01-01

    Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular nucleotides on ion transport in the thick ascending limb (TAL). In the medullary thick ascending limb (mTAL), basolateral P2X receptors markedly (~25%) inhibit NaCl absorption. Although this segment does express both apical and basolateral P2Y2 receptors, acute activation of the basolateral P2Y2 receptors had no apparent effect on transepithelial ion transport. Here we studied, if the absence of the P2Y2 receptor causes chronic alterations in mTAL NaCl absorption by comparing basal and AVP-stimulated transepithelial transport rates. We used perfused mouse mTALs to electrically measure NaCl absorption in juvenile (<35 days) and adult (>35 days) male mice. Using microelectrodes, we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and thus, transepithelial NaCl absorption (equivalent short circuit current, I'sc). We find that mTALs from adult wild type (WT) mice have significantly lower NaCl absorption rates when compared to mTALs from juvenile WT mice. This could be attributed to significantly higher Rtevalues in mTALs from adult WT mice. This pattern was not observed in mTALs from P2Y2 receptor knockout (KO) mice. In addition, adult P2Y2 receptor KO mTALs have significantly lower Vtevalues compared to the juvenile. No difference in absolute I'sc was observed when comparing mTALs from WT and KO mice. AVP stimulated the mTALs to similar increases of NaCl absorption irrespective of the absence of the P2Y2 receptor. No difference was observed in the medullary expression level of NKCC2 in between the genotypes. These data indicate that the lack of P2Y2 receptors does not cause substantial differences in resting and AVP-stimulated NaCl absorption

  8. Small Conductance Ca2+-Activated K+ Channel Knock-Out Mice Reveal the Identity of Calcium-Dependent Afterhyperpolarization Currents

    PubMed Central

    Bond, Chris T.; Herson, Paco S.; Strassmaier, Timothy; Hammond, Rebecca; Stackman, Robert; Maylie, James; Adelman, John P.

    2010-01-01

    Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+ channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+(SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP. PMID:15190101

  9. Reduced Expression of P2Y2 Receptor and Acetylcholinesterase at Neuromuscular Junction of P2Y1 Receptor Knock-out Mice.

    PubMed

    Xu, Miranda L; Bi, Cathy W C; Cheng, Lily K W; Mak, Shinghung; Yao, Ping; Luk, Wilson K W; Lau, Kitty K M; Cheng, Anthony W M; Tsim, Karl W K

    2015-11-01

    ATP is co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 and P2Y2 receptors in the muscle regulated post-synaptic gene expressions. To further support the notion that P2Y receptors are playing indispensable role in formation of post-synaptic specifications at the nmj, the knock-out mice of P2Y1 receptor (P2Y1R (-/-)) were employed here for analyses. In P2Y1R (-/-) mice, the expression of P2Y2 receptor in muscle was reduced by over 50 %, as compared to P2Y1R (+/+) mice. In parallel, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased. In the analysis of the expression of anchoring subunits of AChE in P2Y1R (-/-) mice, the proline-rich membrane anchor (PRiMA) subunit was reduced by 60 %; while the collagen tail (ColQ) subunit was reduced by 50 %. AChE molecular forms in the muscle were not changed, except the amount of enzyme was reduced. Immuno-staining of P2Y1R (-/-) mice nmj, both AChE and AChR were still co-localized at the nmj, and the staining was diminished. Taken together our data demonstrated that P2Y1 receptor regulated the nmj gene expression.

  10. Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice

    PubMed Central

    Vicidomini, Cinzia; Ponzoni, Luisa; Lim, Dmitry; Schmeisser, Michael; Reim, Dominik; Morello, Noemi; Orelanna, Daniel; Tozzi, Alessandro; Durante, Valentina; Scalmani, Paolo; Mantegazza, Massimo; Genazzani, Armando A.; Giustetto, Maurizio; Sala, Mariaelvina; Calabresi, Paolo; Boeckers, Tobias M.; Sala, Carlo; Verpelli, Chiara

    2016-01-01

    SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with Autism Spectrum disorders ASD, and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11-/- mice in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating mGlu5 receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5 receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) ameliorated the functional and behavioral defects that were observed in Shank3Δ11-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations. PMID:27021819

  11. Dietary cladode powder from wild type and domesticated Opuntia species reduces atherogenesis in apoE knock-out mice.

    PubMed

    Garoby-Salom, Sandra; Guéraud, Françoise; Camaré, Caroline; de la Rosa, Ana-Paulina Barba; Rossignol, Michel; Santos Díaz, María del Socorro; Salvayre, Robert; Negre-Salvayre, Anne

    2016-03-01

    Dietary intake of Opuntia species may prevent the development of cardiovascular diseases. The present study was designed to characterize the biological antioxidant and anti-inflammatory properties of Opuntia species and to investigate whether Opuntia cladodes prevent the development of atherosclerosis in vivo, in apoE(-)KO mice. The effects of the two Opuntia species, the wild Opuntia streptacantha and the domesticated Opuntia ficus-indica, were tested on the generation of intra- and extracellular reactive oxygen species (ROS) production and kinetics of the LDL oxidation by murine CRL2181 endothelial cells and on the subsequent inflammatory signaling leading to the adhesion of monocytes on the activated endothelium and the formation of foam cells. Opuntia species blocked the extracellular ROS (superoxide anion) generation and LDL oxidation by CRL2181, as well as the intracellular ROS rise and signaling evoked by the oxidized LDL, including the nuclear translocation of the transcription factor NFκB, the expression of ICAM-1 and VCAM-1 adhesion molecules, and the adhesion of monocytes to CRL2181. In vivo, Opuntia significantly reduced the formation of atherosclerotic lesions and the accumulation of 4-hydroxynonenal adducts in the vascular wall of apoE-KO mice, indicating that Opuntia cladodes prevent lipid oxidation in the vascular wall. In conclusion, wild and domesticated Opuntia species exhibit antioxidant, anti-inflammatory, and antiatherogenic properties which emphasize their nutritional benefit for preventing cardiovascular diseases.

  12. Genetic-background modulation of core and variable autistic-like symptoms in Fmr1 knock-out mice.

    PubMed

    Pietropaolo, Susanna; Guilleminot, Aurélie; Martin, Benoît; D'Amato, Francesca R; Crusio, Wim E

    2011-02-22

    No animal models of autism spectrum disorders (ASD) with good construct validity are currently available; using genetic models of pathologies characterized by ASD-like deficits, but with known causes, may be therefore a promising strategy. The Fmr1-KO mouse is an example of this approach, modeling Fragile X syndrome, a well-known genetic disorder presenting ASD symptoms. The Fmr1-KO is available on different genetic backgrounds (FVB versus C57BL/6), which may explain some of the conflicting results that have been obtained with these mutants up till now. Fmr1 KO and their wild-type littermates on both the FVB and C57BL/6 genetic backgrounds were examined on a battery of tests modeling the clinical symptoms of ASD, including the triad of core symptoms (alterations in social interaction and communication, presence of repetitive behaviors), as well as the secondary symptoms (disturbances in sensori-motor reactivity and in circadian patterns of activity, epileptic events). Fmr1-KO mice displayed autistic-like core symptoms of altered social interaction and occurrence of repetitive behaviors with additional hyperactivity. The genetic background modulated the effects of the Fmr1 deletion and it appears that the C57BL/6 background may be more suitable for further research on core autistic-like symptoms. The Fmr1-mouse line does not recapitulate all of the main core and secondary ASD symptoms, but still can be useful to elucidate the neurobiological mechanisms underlying specific ASD-like endophenotypes.

  13. Genetic-Background Modulation of Core and Variable Autistic-Like Symptoms in Fmr1 Knock-Out Mice

    PubMed Central

    Pietropaolo, Susanna; Guilleminot, Aurélie; Martin, Benoît; D'Amato, Francesca R.; Crusio, Wim E.

    2011-01-01

    Background No animal models of autism spectrum disorders (ASD) with good construct validity are currently available; using genetic models of pathologies characterized by ASD-like deficits, but with known causes, may be therefore a promising strategy. The Fmr1-KO mouse is an example of this approach, modeling Fragile X syndrome, a well-known genetic disorder presenting ASD symptoms. The Fmr1-KO is available on different genetic backgrounds (FVB versus C57BL/6), which may explain some of the conflicting results that have been obtained with these mutants up till now. Methods Fmr1 KO and their wild-type littermates on both the FVB and C57BL/6 genetic backgrounds were examined on a battery of tests modeling the clinical symptoms of ASD, including the triad of core symptoms (alterations in social interaction and communication, presence of repetitive behaviors), as well as the secondary symptoms (disturbances in sensori-motor reactivity and in circadian patterns of activity, epileptic events). Results Fmr1-KO mice displayed autistic-like core symptoms of altered social interaction and occurrence of repetitive behaviors with additional hyperactivity. The genetic background modulated the effects of the Fmr1 deletion and it appears that the C57BL/6 background may be more suitable for further research on core autistic-like symptoms. Conclusions The Fmr1-mouse line does not recapitulate all of the main core and secondary ASD symptoms, but still can be useful to elucidate the neurobiological mechanisms underlying specific ASD-like endophenotypes. PMID:21364941

  14. A role for glucocorticoid-signaling in depression-like behavior of gastrin-releasing peptide receptor knock-out mice.

    PubMed

    Monje, Francisco J; Kim, Eun-Jung; Cabatic, Maureen; Lubec, Gert; Herkner, Kurt R; Pollak, Daniela D

    2011-08-01

    Abstract Background. The gastrin-releasing peptide receptor (GRPR) is highly expressed in the limbic system, where it importantly regulates emotional functions and in the suprachiasmatic nucleus, where it is central for the photic resetting of the circadian clock. Mice lacking GRPR presented with deficient light-induced phase shift in activity as well altered emotional learning and amygdala function. The effect of GRPR deletion on depression-like behavior and its molecular signature in the amygdala, however, has not yet been evaluated. Methods. GRPR knock-out mice (GRPR-KO) were tested in the forced-swim test and the sucrose preference test for depression-like behavior. Gene expression in the basolateral nucleus of the amygdala was evaluated by micorarray analysis subsequent to laser-capture microdissection-assisted extraction of mRNA. The expression of selected genes was confirmed by RT-PCR. Results. GRPR-KO mice were found to present with increased depression-like behavior. Microarray analysis revealed down-regulation of several glucocorticoid-responsive genes in the basolateral amygdala. Acute administration of dexamethasone reversed the behavioral phenotype and alterations in gene expression. Discussion. We propose that deletion of GRPR leads to the induction of depression-like behavior which is paralleled by dysregulation of amygdala gene expression, potentially resulting from deficient light-induced corticosterone release in GRPR-KO.

  15. [Effect of CD40 knock out on cytotoxic effector function in CD8(+) T cell of mice with cigarette smoke-induced emphysema].

    PubMed

    Wang, Q; Deng, T T; Kuang, L J; Qiu, S L; Liang, Y; Zhong, X N; He, Z Y; Zhang, J Q; Bai, J; Li, M H

    2016-05-31

    To explore the effect of CD40 knock out on the cytotoxic function of CD8(+) T cell of mice with cigarette smoke-induced emphysema. A total of 40 male C57 mice were divided into four groups according to the random number table, including CD40(+ /+) control group, CD40(+ /+) smoke-exposure group, CD40(-/-)control group, CD40(-/-)smoke-exposure group. The smoke-exposure groups were exposed to cigarette smoke for 24 weeks to establish emphysema model. Morphological changes were evaluated by linear intercepts. The percentages of CD8, perforin, granzyme B positive cells were evaluated by immunohistochemistry. The mRNA expressions of perforin, granzyme B, interleukin (IL) -27 were measured by fluorescent real time quantitative polymerase chain reaction (RT-PCR). The IL-27 cytokine level was tested by enzyme-linked immunosorbent assay (ELISA). The mean linear intercepts in CD40(+ /+) smoke-exposure group was significantly higher than CD40(+ /+) control group, CD40(-/-)control group, and CD40(-/-)smoke-exposure group [(37.2±3.6) vs (24.0±3.4), (22.5±2.4), (29.9±1.7) μm] (all P<0.05). CD40(-/-)smoke-exposure group was higher than CD40(+ /+) control group, CD40(-/-)control group (all P<0.05). The percentages of CD8 positive, perforin positive and granzyme B positive cells in CD40(+ /+) smoke-exposure group [(16.3±2.3)%, (11.4±2.1)%, (10.7±1.9)%] were significantly higher than CD40(+ /+) control group [(8.3±1.6)%, (5.1±1.2)%, (4.6±1.0)%], CD40(-/-)control group [ (6.4±1.5)%, (4.3±1.0)%, (4.2±1.0)%] and CD40(-/-)smoke-exposure group [(8.6±1.7)%, (5.6±1.3)%, (5.5±1.3)%] (all P<0.05). RT-PCR results showed that the mRNA expressions of perforin, granzyme B and IL-27 in CD40(+ /+) smoke-exposure group [(20.3±7.3), (18.3±12.3), (2.2±0.7)] were significantly higher than CD40(+ /+) control group [(9.4±4.8), (10.6±3.8), (1.3±0.6)], CD40(-/-)control group [ (8.1±3.1), (7.7±3.5), (1.1±0.5)] and CD40(-/-)smoke-exposure group [(12.9±6.2), (10.4±4.6), (1.5±0

  16. Antidepressant activity: contribution of brain microdialysis in knock-out mice to the understanding of BDNF/5-HT transporter/5-HT autoreceptor interactions

    PubMed Central

    Gardier, Alain M.

    2013-01-01

    Why antidepressants vary in terms of efficacy is currently unclear. Despite the leadership of selective serotonin reuptake inhibitors (SSRIs) in the treatment of depression, the precise neurobiological mechanisms involved in their therapeutic action are poorly understood. A better knowledge of molecular interactions between monoaminergic system, pre- and post-synaptic partners, brain neuronal circuits and regions involved may help to overcome limitations of current treatments and identify new therapeutic targets. Intracerebral in vivo microdialysis (ICM) already provided important information about the brain mechanism of action of antidepressants first in anesthetized rats in the early 1990s, and since then in conscious wild-type or knock-out mice. The principle of ICM is based on the balance between release of neurotransmitters (e.g., monoamines) and reuptake by selective transporters [e.g., serotonin transporter for serotonin 5-hydroxytryptamine (5-HT)]. Complementary to electrophysiology, this technique reflects pre-synaptic monoamines release and intrasynaptic events corresponding to ≈80% of whole brain tissue content. The inhibitory role of serotonergic autoreceptors infers that they limit somatodendritic and nerve terminal 5-HT release. It has been proposed that activation of 5-HT1A and 5-HT1B receptor sub-types limits the antidepressant-like activity of SSRIs. This hypothesis is based partially on results obtained in ICM experiments performed in naïve, non-stressed rodents. The present review will first remind the principle and methodology of ICM performed in mice. The crucial need of developing animal models that display anxiety and depression-like behaviors, neurochemical and brain morphological phenotypes reminiscent of these mood disorders in humans, will be underlined. Recently developed genetic mouse models have been generated to independently manipulate 5-HT1A auto and heteroreceptors and ICM helped to clarify the role of the pre-synaptic component

  17. Circadian rhythms in heart rate, motility, and body temperature of wild-type C57 and eNOS knock-out mice under light-dark, free-run, and after time zone transition.

    PubMed

    Arraj, M; Lemmer, B

    2006-01-01

    The nitric oxide (NO) system is involved in the regulation of the cardiovascular system in controlling central and peripheral vascular tone and cardiac functions. It was the aim of this study to investigate in wild-type C57BL/6 and endothelial nitric oxide synthase (eNOS) knock-out mice (eNOS-/-) the contribution of NO on the circadian rhythms in heart rate (HR), motility (motor activity [MA]), and body temperature (BT) under various environmental conditions. Experiments were performed in 12:12 h of a light:dark cycle (LD), under free-run in total darkness (DD), and after a phase delay shift of the LD cycle by -6 h (i.e., under simulation of a westward time zone transition). All parameters were monitored by radiotelemetry in freely moving mice. In LD, no significant differences in the rhythms of HR and MA were observed between the two strains of mice. BT, however, was significantly lower during the light phase in eNOS-/- mice, resulting in a significantly greater amplitude. The period of the free-running rhythm in DD was slightly shorter for all variables, though not significant. In general, rhythmicity was greater in eNOS-/- than in C57 mice both in LD and DD. After a delay shift of the LD cycle, HR and BT were resynchronized to the new LD schedule within 5-6 days, and resynchronization of MA occurred within 2-3 days. The results in telemetrically instrumented mice show that complete knock-out of the endothelial NO system--though expressed in the suprachiasmatic nuclei and in peripheral tissues--did not affect the circadian organization of heart rate and motility. The circadian regulation of the body temperature was slightly affected in eNOS-/- mice.

  18. Conversion of the Modulatory Actions of Dopamine on Spinal Reflexes from Depression to Facilitation in D3 Receptor Knock-Out Mice

    PubMed Central

    Clemens, Stefan; Hochman, Shawn

    2009-01-01

    Descending monoaminergic systems modulate spinal cord function, yet spinal dopaminergic actions are poorly understood. Using the in vitro lumbar cord, we studied the effects of dopamine and D2-like receptor ligands on spinal reflexes in wild-type (WT) and D3-receptor knock-out mice (D3KO). Low dopamine levels (1µm) decreased the monosynaptic “stretch” reflex (MSR) amplitude in WT animals and increased it in D3KO animals. Higher dopamine concentrations (10 –100µm) decreased MSR amplitudes in both groups, but always more strongly in WT. Like low dopamine, the D3 receptor agonists pergolide and PD 128907 reduced MSR amplitude in WT but not D3KO mice. Conversely, D3 receptor antagonists (GR 103691 and nafadotride) increased the MSR in WT but not in D3KO mice. In comparison,D2-preferring agonists bromocriptine and quinpirole depressed the MSR in both groups. Low dopamine (1–5 µm) also depressed longer-latency (presumably polysynaptic) reflexes in WT but facilitated responses in D3KO mice. Additionally, in some experiments (e.g., during 10 µm dopamine or pergolide in WT), polysynaptic reflexes were facilitated in parallel to MSR depression, demonstrating differential modulatory control of these reflex circuits. Thus, low dopamine activates D3 receptors to limit reflex excitability. Moreover, in D3 ligand-insensitive mice, excitatory actions are unmasked, functionally converting the modulatory action of dopamine from depression to facilitation. Restless legs syndrome (RLS) is a CNS disorder involving abnormal limb sensations. Because RLS symptoms peak at night when dopamine levels are lowest, are relieved by D3 agonists, and likely involve increased reflex excitability, the D3KO mouse putatively explains how impaired D3 activity could contribute to this sleep disorder. PMID:15601940

  19. Conversion of the modulatory actions of dopamine on spinal reflexes from depression to facilitation in D3 receptor knock-out mice.

    PubMed

    Clemens, Stefan; Hochman, Shawn

    2004-12-15

    Descending monoaminergic systems modulate spinal cord function, yet spinal dopaminergic actions are poorly understood. Using the in vitro lumbar cord, we studied the effects of dopamine and D2-like receptor ligands on spinal reflexes in wild-type (WT) and D3-receptor knock-out mice (D3KO). Low dopamine levels (1 microM) decreased the monosynaptic "stretch" reflex (MSR) amplitude in WT animals and increased it in D3KO animals. Higher dopamine concentrations (10-100 microM) decreased MSR amplitudes in both groups, but always more strongly in WT. Like low dopamine, the D3 receptor agonists pergolide and PD 128907 reduced MSR amplitude in WT but not D3KO mice. Conversely, D3 receptor antagonists (GR 103691 and nafadotride) increased the MSR in WT but not in D3KO mice. In comparison, D2-preferring agonists bromocriptine and quinpirole depressed the MSR in both groups. Low dopamine (1-5 microM) also depressed longer-latency (presumably polysynaptic) reflexes in WT but facilitated responses in D3KO mice. Additionally, in some experiments (e.g., during 10 microM dopamine or pergolide in WT), polysynaptic reflexes were facilitated in parallel to MSR depression, demonstrating differential modulatory control of these reflex circuits. Thus, low dopamine activates D3 receptors to limit reflex excitability. Moreover, in D3 ligand-insensitive mice, excitatory actions are unmasked, functionally converting the modulatory action of dopamine from depression to facilitation. Restless legs syndrome (RLS) is a CNS disorder involving abnormal limb sensations. Because RLS symptoms peak at night when dopamine levels are lowest, are relieved by D3 agonists, and likely involve increased reflex excitability, the D3KO mouse putatively explains how impaired D3 activity could contribute to this sleep disorder.

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

    PubMed

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

    2017-07-07

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

  1. Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.

    PubMed

    Zhu, Xuewei; Lee, Ji-Young; Timmins, Jenelle M; Brown, J Mark; Boudyguina, Elena; Mulya, Anny; Gebre, Abraham K; Willingham, Mark C; Hiltbold, Elizabeth M; Mishra, Nilamadhab; Maeda, Nobuyo; Parks, John S

    2008-08-22

    Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.

  2. Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression.

    PubMed

    Gao, Congxiao; Maeno, Toshitaka; Ota, Fumi; Ueno, Manabu; Korekane, Hiroaki; Takamatsu, Shinji; Shirato, Ken; Matsumoto, Akio; Kobayashi, Satoshi; Yoshida, Keiichi; Kitazume, Shinobu; Ohtsubo, Kazuaki; Betsuyaku, Tomoko; Taniguchi, Naoyuki

    2012-05-11

    We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

  3. Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis

    PubMed Central

    Bhattacharya, Parna; Dey, Ranadhir; Dagur, Pradeep K.; Joshi, Amritanshu B.; Ismail, Nevien; Gannavaram, Sreenivas; Debrabant, Alain; Akue, Adovi D.; KuKuruga, Mark A.; Selvapandiyan, Angamuthu; McCoy, John Philip; Nakhasi, Hira L.

    2016-01-01

    Background Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. Methodology Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells

  4. Working Memory Impairment in Calcineurin Knock-out Mice Is Associated with Alterations in Synaptic Vesicle Cycling and Disruption of High-Frequency Synaptic and Network Activity in Prefrontal Cortex

    PubMed Central

    Cottrell, Jeffrey R.; Levenson, Jonathan M.; Kim, Sung Hyun; Gibson, Helen E.; Richardson, Kristen A.; Sivula, Michael; Li, Bing; Ashford, Crystle J.; Heindl, Karen A.; Babcock, Ryan J.; Rose, David M.; Hempel, Chris M.; Wiig, Kjesten A.; Laeng, Pascal; Levin, Margaret E.; Ryan, Timothy A.

    2013-01-01

    Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia. PMID:23825400

  5. Knock out of S1P3 receptor signaling attenuates inflammation and fibrosis in bleomycin-induced lung injury mice model.

    PubMed

    Murakami, Ken; Kohno, Masataka; Kadoya, Masatoshi; Nagahara, Hidetake; Fujii, Wataru; Seno, Takahiro; Yamamoto, Aihiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Morita, Satoshi; Nakada, Hiroshi; Hla, Timothy; Kawahito, Yutaka

    2014-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein-coupled receptors (S1P1-5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.

  6. Effects of Ascorbic Acid on Carcinogenicity and Acute Toxicity of Nickel Subsulfide, and on Tumor Transplants Growth in Gulonolactone Oxidase Knock-Out Mice and Wild-type C57BL Mice

    PubMed Central

    Kasprzak, Kazimierz S.; Diwan, Bhalchandra A.; Kaczmarek, Monika Z.; Logsdon, Daniel L.; Fivash, Mathew J.; Salnikow, Konstantin

    2011-01-01

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono--lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni3S2), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni3S2 carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni3S2. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni3S2 in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni3S2 carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni3S2 and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies. PMID:21878346

  7. Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice.

    PubMed

    Kasprzak, Kazimierz S; Diwan, Bhalchandra A; Kaczmarek, Monika Z; Logsdon, Daniel L; Fivash, Mathew J; Salnikow, Konstantin

    2011-11-15

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono--lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni₃S₂), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni₃S₂ carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni₃S₂. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni₃S₂ in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni₃S₂ carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni₃S₂ and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies.

  8. Involvement of mitogen-activated protein kinases (MAPKs) during testicular ischemia-reperfusion injury in nuclear factor-kappaB knock-out mice.

    PubMed

    Minutoli, Letteria; Antonuccio, Pietro; Polito, Francesca; Bitto, Alessandra; Fiumara, Tiziana; Squadrito, Francesco; Nicotina, Piero Antonio; Arena, Salvatore; Marini, Herbert; Romeo, Carmelo; Altavilla, Domenica

    2007-07-12

    Nuclear factor kappa-B (NF-kappaB), extracellular regulated kinase (ERK 1/2) and c-jun-N terminal kinase (JNK) play an important role in testicular ischemia. We investigated the patterns of ERK1/2, JNK and p38 activation in NF-kappaB knockout (KO) mice subjected to testicular torsion. KO and normal littermate wild-type (WT) animals underwent at 1 h testicular ischemia followed by 24 h reperfusion (TI/R). Sham testicular ischemia-reperfusion mice served as controls. ERK 1/2, JNK and p38 expression by western blot analysis, tumor necrosis factor-alpha (TNF-alpha) expression (RT-PCR and western blot analysis) and a complete histological examination were carried out. TI/R caused a greater increase in phosphorylated form of ERK 1/2 in KO mice than in WT animals in either the ischemic testis and the contralateral one. By contrary, active form of JNK and p38 were completely abrogated in both testes of KO mice, while WT animals showed a significant activation of those kinases in both testes. TNF-alpha expression was markedly reduced in KO mice when compared to WT mice either at the mRNA and the protein level. Finally TI/R-induced histological damage was markedly reduced in KO mice. Our data indicate that NF-kappaB plays a pivotal role in the development of testicular ischemia-reperfusion injury and suggest that, in the absence of the transcriptional factor, the up-stream signal JNK and p38 may be abrogated while ERK 1/2 activity is enhanced.

  9. Unexpected Lack of Hypersensitivity in LRRK2 Knock-out Mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

    PubMed Central

    Andres-Mateos, Eva; Mejias, Rebeca; Sasaki, Masayuki; Li, Xiaojie; Lin, Brian M; Biskup, Saskia; Zhang, Li; Banerjee, Rebecca; Thomas, Bobby; Yang, Lichuan; Liu, Guosheng; Beal, M Flint; Huso, David L; Dawson, Ted M; Dawson, Valina L

    2010-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinson's disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knockout (KO) mice lacking the kinase domain of LRRK2. Here we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild type (WT) mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP. PMID:20016100

  10. Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-06-01

    The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

  11. Gene expression and mRNA editing of serotonin receptor 2C in brains of HPRT gene knock-out mice, an animal model of Lesch-Nyhan disease

    PubMed Central

    Bertelli, Matteo; Alushi, Brunilda; Veicsteinas, Arsenio; Jinnah, H.A.; Micheli, Vanna

    2016-01-01

    Lesch-Nyhan disease (LND), a genetic disorder associated with motor and psychiatric disturbance and self-injurious behaviour (SIB) is caused by a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). The connection between enzyme deficiency and neurological involvement is still unclear. Evidence exists for a role of basal ganglia dysfunction with decreased dopamine and excess serotonin striatal content. In this study, we investigate the role of serotonin receptor 2C (HTR2C) in the brains of HPRT gene knock-out mice, a model of LND. HTR2C expression is analyzed by real-time polymerase chain reaction (PCR) using SYBR-green detection methods. The percentage of edited HTR2C mRNA was determined by direct sequencing of amplification products of the region containing the editing sites. We found a 55% increase in the expression of HTR2C gene but no significant difference in mRNA editing levels between knock-out and control mice. The above alteration found in HPRT-deficient mice is similar to those found in other animal models used to study aggressive and self-injurious behaviour. PMID:19473847

  12. 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

    2016-05-04

    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.

  13. Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice

    SciTech Connect

    Kasprzak, Kazimierz S.; Diwan, Bhalchandra A.; Kaczmarek, Monika Z.; Logsdon, Daniel L.; Fivash, Mathew J.; Salnikow, Konstantin

    2011-11-15

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono- < gamma > -lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni{sub 3}S{sub 2}), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni{sub 3}S{sub 2} carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni{sub 3}S{sub 2}. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni{sub 3}S{sub 2} in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni{sub 3}S{sub 2} carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni{sub 3}S{sub 2} and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies. -- Highlights: Black-Right-Pointing-Pointer Ascorbate depletion enhances carcinogenicity and acute toxicity of nickel. Black-Right-Pointing-Pointer Gulo-/- mice unable to synthesize ascorbate were used in this study. Black

  14. Gonadotropin-releasing hormone receptor (Gnrhr) gene knock out: Normal growth and development of sensory, motor and spatial orientation behavior but altered metabolism in neonatal and prepubertal mice

    PubMed Central

    Busby, Ellen R.; Sherwood, Nancy M.

    2017-01-01

    Gonadotropin-releasing hormone (GnRH) is important in the control of reproduction, but its actions in non-reproductive processes are less well known. In this study we examined the effect of disrupting the GnRH receptor in mice to determine if growth, metabolism or behaviors that are not associated with reproduction were affected. To minimize the effects of other hormones such as FSH, LH and sex steroids, the neonatal-prepubertal period of 2 to 28 days of age was selected. The study shows that regardless of sex or phenotype in the Gnrhr gene knockout line, there was no significant difference in the daily development of motor control, sensory detection or spatial orientation among the wildtype, heterozygous or null mice. This included a series of behavioral tests for touch, vision, hearing, spatial orientation, locomotory behavior and muscle strength. Neither the daily body weight nor the final weight on day 28 of the kidney, liver and thymus relative to body weight varied significantly in any group. However by day 28, metabolic changes in the GnRH null females compared with wildtype females showed a significant reduction in inguinal fat pad weight normalized to body weight; this was accompanied by an increase in glucose compared with wildtype females shown by Student-Newman-Keuls Multiple Comparison test and Student's unpaired t tests. Our studies show that the GnRH-GnRHR system is not essential for growth or motor/sensory/orientation behavior during the first month of life prior to puberty onset. The lack of the GnRH-GnRHR axis, however, did affect females resulting in reduced subcutaneous inguinal fat pad weight and increased glucose with possible insulin resistance; the loss of the normal rise of estradiol at postnatal days 15–28 may account for the altered metabolism in the prepubertal female pups. PMID:28346489

  15. Gonadotropin-releasing hormone receptor (Gnrhr) gene knock out: Normal growth and development of sensory, motor and spatial orientation behavior but altered metabolism in neonatal and prepubertal mice.

    PubMed

    Busby, Ellen R; Sherwood, Nancy M

    2017-01-01

    Gonadotropin-releasing hormone (GnRH) is important in the control of reproduction, but its actions in non-reproductive processes are less well known. In this study we examined the effect of disrupting the GnRH receptor in mice to determine if growth, metabolism or behaviors that are not associated with reproduction were affected. To minimize the effects of other hormones such as FSH, LH and sex steroids, the neonatal-prepubertal period of 2 to 28 days of age was selected. The study shows that regardless of sex or phenotype in the Gnrhr gene knockout line, there was no significant difference in the daily development of motor control, sensory detection or spatial orientation among the wildtype, heterozygous or null mice. This included a series of behavioral tests for touch, vision, hearing, spatial orientation, locomotory behavior and muscle strength. Neither the daily body weight nor the final weight on day 28 of the kidney, liver and thymus relative to body weight varied significantly in any group. However by day 28, metabolic changes in the GnRH null females compared with wildtype females showed a significant reduction in inguinal fat pad weight normalized to body weight; this was accompanied by an increase in glucose compared with wildtype females shown by Student-Newman-Keuls Multiple Comparison test and Student's unpaired t tests. Our studies show that the GnRH-GnRHR system is not essential for growth or motor/sensory/orientation behavior during the first month of life prior to puberty onset. The lack of the GnRH-GnRHR axis, however, did affect females resulting in reduced subcutaneous inguinal fat pad weight and increased glucose with possible insulin resistance; the loss of the normal rise of estradiol at postnatal days 15-28 may account for the altered metabolism in the prepubertal female pups.

  16. Meperidine, remifentanil and tramadol but not sufentanil interact with alpha(2)-adrenoceptors in alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knock out mice brain.

    PubMed

    Höcker, Jan; Weber, Bernd; Tonner, Peter H; Scholz, Jens; Brand, Philipp-Alexander; Ohnesorge, Henning; Bein, Berthold

    2008-03-17

    alpha(2)-adrenoceptor agonists like clonidine or dexmedetomidine increase the sedative and analgesic actions of opioids. Furthermore opioids like meperidine show potent anti-shivering effects like alpha(2)-adrenoceptor agonists. The underlying molecular mechanisms of these effects are still poorly defined. The authors therefore studied the ability of four different opioids (meperidine, remifentanil, sufentanil and tramadol) to interact with different alpha(2)-adrenoceptor subtypes in mice lacking individual alpha(2A)-, alpha(2B)- or alpha(2C)-adrenoceptors (alpha(2)-adrenoceptor knock out (alpha(2)-AR KO) mice)). The interaction of opioids with alpha(2)-adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of alpha(2A)-, alpha(2B)- or alpha(2C)-adrenoceptor deficient mice. Displacement of the radiolabelled alpha(2)-adrenoceptor agonist [(125)I]-paraiodoclonidine ([(125)I]-PIC) from alpha(2)-adrenoceptors in different brain regions by increasing opioid concentrations was measured, and binding affinity of the analysed opioids to alpha(2)-adrenoceptor subtypes in different brain regions was quantified. Meperidine, remifentanil and tramadol but not sufentanil provoked dose dependent displacement of specifically bound [(125)I]-PIC from all alpha(2)-adrenoceptor subtypes in cortex, cerebellum, medulla oblongata, thalamus, hippocampus and pons. Required concentrations of meperidine and remifentanil for [(125)I]-PIC displacement from alpha(2B)- and alpha(2C)-adrenoceptors were lower than from alpha(2A)-adrenoceptors, indicating higher binding affinity for alpha(2B)- and alpha(2C)-adrenoceptors. In contrast, [(125)I]-PIC displacement by tramadol indicated higher binding affinity to alpha(2A)-adrenoceptors than to alpha(2B)- and alpha(2C)-adrenoceptors. Our results indicate that meperidine, remifentanil and tramadol interact with alpha(2)-adrenoceptors in mouse brain showing different affinity for alpha(2A)-, alpha(2B)- and alpha(2C

  17. Susceptibility of T cell receptor-α chain knock-out mice to ultraviolet B light and fluorouracil: a novel model for drug-induced cutaneous lupus erythematosus

    PubMed Central

    YOSHIMASU, T; NISHIDE, T; SEO, N; HIROI, A; OHTANI, T; UEDE, K; FURUKAWA, F

    2004-01-01

    The anticancer agent 5-fluorouracil (FU) frequently induces cutaneous lupus erythematosus (LE) lesions on sun exposed sites. Based on this observation, we have tried to establish a cutaneous LE model of C57BL/6 J (B6) mice, B6 T cell receptor (TCR)-α–/– mice and B6 TCR-δ–/– mice treated with FU and/or ultraviolet B light (UVBL) in order to clarify the role of T cells and the cytokine profile of cutaneous lupus lesions. Cutaneous LE-like skin lesions could be induced in TCR-α–/– mice with low FU (0·2 mg) plus UVBL, and in B6 mice treated with a high dose of FU (2·0 mg) plus UVBL. In contrast, low FU plus UVBL induced such skin lesions in TCR-δ–/– mice at a very low incidence. Specifically, the skin lesions of TCR-α–/– mice with low FU plus UVBL appeared more rapidly and were more severe than lesions in B6 mice. The former had the common characteristic features of human chronic cutaneous LE such as typical histology, positive IgG at the dermoepidermal junction, low antinuclear antibody and low mortality. Furthermore, a Th1 response was induced in the development of drug-induced cutaneous LE. FU and UVBL-induced cutaneous LE-like eruption is an excellent model for better understanding the pathomechanisms of skin lesion development in LE. PMID:15086387

  18. Attrition of Hepatic Damage Inflicted by Angiotensin II with α-Tocopherol and β-Carotene in Experimental Apolipoprotein E Knock-out Mice.

    PubMed

    Gopal, Kaliappan; Gowtham, Munusamy; Sachin, Singh; Ravishankar Ram, Mani; Shankar, Esaki M; Kamarul, Tunku

    2015-12-16

    Angiotensin II is one of the key regulatory peptides implicated in the pathogenesis of liver disease. The mechanisms underlying the salubrious role of α-tocopherol and β-carotene on liver pathology have not been comprehensively assessed. Here, we investigated the mechanisms underlying the role of Angiotensin II on hepatic damage and if α-tocopherol and β-carotene supplementation attenuates hepatic damage. Hepatic damage was induced in Apoe(-/-)mice by infusion of Angiotensin II followed by oral administration with α-tocopherol and β-carotene-enriched diet for 60 days. Investigations showed fibrosis, kupffer cell hyperplasia, hepatocyte degeneration and hepatic cell apoptosis; sinusoidal dilatation along with haemorrhages; evidence of fluid accumulation; increased ROS level and increased AST and ALT activities. In addition, tPA and uPA were down-regulated due to 42-fold up-regulation of PAI-1. MMP-2, MMP-9, MMP-12, and M-CSF were down-regulated in Angiotensin II-treated animals. Notably, α-tocopherol and β-carotene treatment controlled ROS, fibrosis, hepatocyte degeneration, kupffer cell hyperplasia, hepatocyte apoptosis, sinusoidal dilatation and fluid accumulation in the liver sinusoids, and liver enzyme levels. In addition, PAI-1, tPA and uPA expressions were markedly controlled by β-carotene treatment. Thus, Angiotensin II markedly influenced hepatic damage possibly by restraining fibrinolytic system. We concluded that α-tocopherol and β-carotene treatment has salubrious role in repairing hepatic pathology.

  19. Metallothionein (MT) 1/2 expression in MT 1/2 and MT 3 knock-out mice and Long-Evans Cinnamon (LEC) rats.

    PubMed

    Nakazato, Kyoumi; Nakajima, Katsuyuki; Nakano, Takamitsu; Kodaira, Tsukasa; Nakayama, Kenji; Satoh, Masahiko; Nagamine, Takeaki

    2012-02-01

    Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies.

  20. Characterization of fast-twitch and slow-twitch skeletal muscles of calsequestrin 2 (CASQ2)-knock out mice: unexpected adaptive changes of fast-twitch muscles only.

    PubMed

    Valle, Giorgia; Vergani, Barbara; Sacchetto, Roberta; Reggiani, Carlo; De Rosa, Edith; Maccatrozzo, Lisa; Nori, Alessandra; Villa, Antonello; Volpe, Pompeo

    2016-12-01

    This study investigates the functional role of calsequestrin 2 (CASQ2) in both fast-twitch and slow-twitch skeletal muscles by using CASQ2-/- mice; CASQ2 is expressed throughout life in slow-twitch muscles, but only in the developmental and neonatal stages in fast-twitch muscles. CASQ2-/- causes increase in calsequestrin 1 (CASQ1) expression, but without functional changes in both muscle types. CASQ2-/- mice have ultrastructural changes in fast-twitch muscles only, i.e., formation of pentads and stacks in the sarcoplasmic reticulum.

  1. Comparative hepatic effects of perfluorooctanoic acid and WY 14,643 in PPARa-knocked out and wild-type mice.

    EPA Science Inventory

    Perfluorooctanoic acid (PFOA) is an environmentally persistent chemical commonly found in humans and wildlife. Induction of liver tumors by PFOA in rodents is thought to be mediated by PPARα activation, although hepatic hypertrophy persists in PPARα-null mice. This study evalua...

  2. Comparative hepatic effects of perfluorooctanoic acid and WY 14,643 in PPARa-knocked out and wild-type mice.

    EPA Science Inventory

    Perfluorooctanoic acid (PFOA) is an environmentally persistent chemical commonly found in humans and wildlife. Induction of liver tumors by PFOA in rodents is thought to be mediated by PPARα activation, although hepatic hypertrophy persists in PPARα-null mice. This study evalua...

  3. Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-β knock-out mice

    PubMed Central

    Matheu, Victor; Treschow, Alexandra; Teige, Ingrid; Navikas, Vaidrius; Issazadeh-Navikas, Shohreh

    2005-01-01

    Background CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-β in the immunomodulatory effects of CpG-ODN is not known. Objective Here, we aimed to elucidate the role of IFN-β in the anti-allergic effect of CpG motifs. Methods We assessed the immune response in OVA-primed/OVA-challenged IFN-β knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment with synthetic CpG motifs. Results Vaccination with CpG-ODN reduced the number of cells in airways of OVA-sensitized WT but not IFN-β-/- mice. Although airway eosinophilia was reduced in both treated groups, they were significantly higher in IFN-β-/- mice. Other inflammatory cells, such as lymphocytes and macrophages were enhanced in airways by CpG treatment in IFN-β-/- mice. The ratio of IFN-γ/IL-4 cytokines in airways was significantly skewed to a Th1 response in WT compared to IFN-β-/- group. In contrast, IL-4 and IgE were reduced with no differences between groups. Ag-specific T-cell proliferation, Th1-cytokines such as IFN-γ, IL-2 and also IL-12 were significantly lower in IFN-β-/- mice. Surprisingly, we discovered that intranasal treatment of mice with CpG-ODN results in mild synovitis particularly in IFN-β-/- mice. Conclusion Our results indicate that induction of Th1 response by therapy with CpG-ODN is only slightly and partially dependent on IFN-β, while IFN-β is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination. PMID:15748290

  4. 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.

  5. Relationships among parvalbumin-immunoreactive neuron density, phase-locked gamma oscillations, and autistic/schizophrenic symptoms in PDGFR-β knock-out and control mice.

    PubMed

    Nakamura, Tomoya; Matsumoto, Jumpei; Takamura, Yusaku; Ishii, Yoko; Sasahara, Masakiyo; Ono, Taketoshi; Nishijo, Hisao

    2015-01-01

    Cognitive deficits and negative symptoms are important therapeutic targets for schizophrenia and autism disorders. Although reduction of phase-locked gamma oscillation has been suggested to be a result of reduced parvalbumin-immunoreactive (putatively, GABAergic) neurons, no direct correlations between these have been established in these disorders. In the present study, we investigated such relationships during pharmacological treatment with a newly synthesized drug, T-817MA, which displays neuroprotective and neurotrophic effects. In this study, we used platelet-derived growth factor receptor-β gene knockout (PDGFR-β KO) mice as an animal model of schizophrenia and autism. These mutant mice display a reduction in social behaviors; deficits in prepulse inhibition (PPI); reduced levels of parvalbumin-immunoreactive neurons in the medical prefrontal cortex, hippocampus, amygdala, and superior colliculus; and a deficit in of auditory phase-locked gamma oscillations. We found that oral administration of T-817MA ameliorated all these symptoms in the PDGFR-β KO mice. Furthermore, phase-locked gamma oscillations were significantly correlated with the density of parvalbumin-immunoreactive neurons, which was, in turn, correlated with PPI and behavioral parameters. These findings suggest that recovery of parvalbumin-immunoreactive neurons by pharmacological intervention relieved the reduction of phase-locked gamma oscillations and, consequently, ameliorated PPI and social behavioral deficits. Thus, our findings suggest that phase-locked gamma oscillations could be a useful physiological biomarker for abnormality of parvalbumin-immunoreactive neurons that may induce cognitive deficits and negative symptoms of schizophrenia and autism, as well as of effective pharmacological interventions in both humans and experimental animals.

  6. Relationships among Parvalbumin-Immunoreactive Neuron Density, Phase-Locked Gamma Oscillations, and Autistic/Schizophrenic Symptoms in PDGFR-β Knock-Out and Control Mice

    PubMed Central

    Nakamura, Tomoya; Matsumoto, Jumpei; Takamura, Yusaku; Ishii, Yoko; Sasahara, Masakiyo; Ono, Taketoshi; Nishijo, Hisao

    2015-01-01

    Cognitive deficits and negative symptoms are important therapeutic targets for schizophrenia and autism disorders. Although reduction of phase-locked gamma oscillation has been suggested to be a result of reduced parvalbumin-immunoreactive (putatively, GABAergic) neurons, no direct correlations between these have been established in these disorders. In the present study, we investigated such relationships during pharmacological treatment with a newly synthesized drug, T-817MA, which displays neuroprotective and neurotrophic effects. In this study, we used platelet-derived growth factor receptor-β gene knockout (PDGFR-β KO) mice as an animal model of schizophrenia and autism. These mutant mice display a reduction in social behaviors; deficits in prepulse inhibition (PPI); reduced levels of parvalbumin-immunoreactive neurons in the medical prefrontal cortex, hippocampus, amygdala, and superior colliculus; and a deficit in of auditory phase-locked gamma oscillations. We found that oral administration of T-817MA ameliorated all these symptoms in the PDGFR-β KO mice. Furthermore, phase-locked gamma oscillations were significantly correlated with the density of parvalbumin-immunoreactive neurons, which was, in turn, correlated with PPI and behavioral parameters. These findings suggest that recovery of parvalbumin-immunoreactive neurons by pharmacological intervention relieved the reduction of phase-locked gamma oscillations and, consequently, ameliorated PPI and social behavioral deficits. Thus, our findings suggest that phase-locked gamma oscillations could be a useful physiological biomarker for abnormality of parvalbumin-immunoreactive neurons that may induce cognitive deficits and negative symptoms of schizophrenia and autism, as well as of effective pharmacological interventions in both humans and experimental animals. PMID:25803852

  7. CRISPR/Cas9-based Pten knock-out and Sleeping Beauty Transposon-mediated Nras knock-in induces hepatocellular carcinoma and hepatic lipid accumulation in mice.

    PubMed

    Gao, Mingming; Liu, Dexi

    2017-07-03

    Both Pten and Nras are downstream mediators of receptor tyrosine kinase activation that plays important roles in controlling cell survival and proliferation. Here, we investigated whether and how Pten loss cross-talks with Nras activation in driving liver cancer development in mice. Somatic disruption of hepatic Pten and overexpression of Nras were achieved in out-bred immunocompetent CD-1 mice through a hydrodynamic delivery of plasmids carrying Sleeping Beauty transposon-based integration of Nras and the CRISPR/Cas9-mediated Pten knockout system. Concurrent Pten knockout and Nras knock-in induced hepatocellular carcinoma, while individual gene manipulation failed. Tumor development was associated with liver fibrosis, hyperlipidemia, hepatic deposition of lipid droplets and glycogen, and hepatomegaly. At the molecular level, lipid droplet formation was primarily contributed by upregulated expression of genes responsible for lipogenesis and fatty acid sequestration, such as Srebpf1, Acc, Pparg and its downstream targets. Our findings demonstrated that Pten disruption was synergized by Nras overexpression in driving hepatocyte malignant transformation, which correlated with extensive formation of lipid droplets.

  8. Depletion of CD4+CD25+ regulatory T cells exacerbates sodium iodide-induced experimental autoimmune thyroiditis in human leucocyte antigen DR3 (DRB1*0301) transgenic class II-knock-out non-obese diabetic mice.

    PubMed

    Flynn, J C; Meroueh, C; Snower, D P; David, C S; Kong, Y M

    2007-03-01

    Both genetic and environmental factors contribute to autoimmune disease development. Previously, we evaluated genetic factors in a humanized mouse model of Hashimoto's thyroiditis (HT) by immunizing human leucocyte antigen DR3 (HLA-DR3) and HLA-DQ8 transgenic class II-knock-out non-obese diabetic (NOD) mice. DR3+ mice were susceptible to experimental autoimmune thyroiditis (EAT) induction by both mouse thyroglobulin (mTg) and human (h) Tg, while DQ8+ mice were weakly susceptible only to hTg. As one environmental factor associated with HT and tested in non-transgenic models is increased sodium iodide (NaI) intake, we examined the susceptibility of DR3+ and/or DQ8+ mice to NaI-induced disease. Mice were treated for 8 weeks with NaI in the drinking water. At 0 x 05% NaI, 23% of DR3+, 0% of DQ8+ and 20% of DR3+DQ8+ mice had thyroid destruction. No spleen cell proliferation to mTg was observed. Most mice had undetectable anti-mTg antibodies, but those with low antibody levels usually had thyroiditis. At 0.3% NaI, a higher percentage of DR3+ and DR3+DQ8+ mice developed destructive thyroiditis, but it was not statistically significant. However, when DR3+ mice had been depleted of CD4+CD25+ regulatory T cells prior to NaI treatment, destructive thyroiditis (68%) and serum anti-mTg antibodies were exacerbated further. The presence of DQ8 molecules does not alter the susceptibility of DR3+DQ8+ mice to NaI-induced thyroiditis, similar to earlier findings with mTg-induced EAT. Susceptibility of DR3+ mice to NaI-induced EAT, in both the presence and absence of regulatory T cells, demonstrates the usefulness of HLA class II transgenic mice in evaluating the roles of environmental factors and immune dysregulation in autoimmune thyroid disease.

  9. Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia*

    PubMed Central

    Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R.; Aranda, Jacob; Grant, Maria B.; Chaqour, Brahim

    2015-01-01

    The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair. PMID:26242736

  10. Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia.

    PubMed

    Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R; Aranda, Jacob; Grant, Maria B; Chaqour, Brahim

    2015-09-18

    The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3'-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.

  11. Hyperactivity of Newborn Pten Knock-out Neurons Results from Increased Excitatory Synaptic Drive

    PubMed Central

    Williams, Michael R.; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T.

    2015-01-01

    Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either “birthdate” or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. PMID:25609613

  12. Dynamics of Sun5 Localization during Spermatogenesis in Wild Type and Dpy19l2 Knock-Out Mice Indicates That Sun5 Is Not Involved in Acrosome Attachment to the Nuclear Envelope

    PubMed Central

    Yassine, Sandra; Escoffier, Jessica; Nahed, Roland Abi; Pierre, Virginie; Karaouzene, Thomas; Ray, Pierre F.; Arnoult, Christophe

    2015-01-01

    The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes. PMID:25775128

  13. Upregulation of the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Axis in the Heart and the Kidney of Growth Hormone Receptor knock-out Mice

    PubMed Central

    GIANI, Jorge F.; MIQUET, Johanna G.; MUÑOZ, Marina C.; BURGHI, Valeria; TOBLLI, Jorge E.; MASTERNAK, Michal M.; KOPCHIC, John J.; BARTKE, Andrzej; TURYN, Daniel; DOMINICI, Fernando P.

    2012-01-01

    Objective Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR−/−) mouse. Design RAS components were evaluated in the heart and the kidney of GHR−/− and control mice by immunohistochemistry and western blotting (n=12 for both groups). Results The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR−/− mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR−/− mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western Blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues. Conclusions The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR−/− mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity. PMID:22947377

  14. Accumulation of Oxidized LDL in the Tendon Tissues of C57BL/6 or Apolipoprotein E Knock-Out Mice That Consume a High Fat Diet: Potential Impact on Tendon Health

    PubMed Central

    Grewal, Navdeep; Thornton, Gail M.; Behzad, Hayedeh; Sharma, Aishwariya; Lu, Alex; Zhang, Peng; Reid, W. Darlene; Granville, David J.; Scott, Alex

    2014-01-01

    Objective Clinical studies have suggested an association between dyslipidemia and tendon injuries or chronic tendon pain; the mechanisms underlying this association are not yet known. The objectives of this study were (1) to evaluate the impact of a high fat diet on the function of load-bearing tendons and on the distribution in tendons of oxidized low density lipoprotein (oxLDL), and (2) to examine the effect of oxLDL on tendon fibroblast proliferation and gene expression. Methods Gene expression (Mmp2, Tgfb1, Col1a1, Col3a1), fat content (Oil Red O staining), oxLDL levels (immunohistochemistry) and tendon biomechanical properties were examined in mice (C57Bl/6 or ApoE -/-) receiving a standard or a high fat diet. Human tendon fibroblast proliferation and gene expression (COL1A1, COL3A1, MMP2) were examined following oxLDL exposure. Results In both types of mice (C57Bl/6 or ApoE -/-), consumption of a high fat diet led to a marked increase in oxLDL deposition in the load-bearing extracellular matrix of the tendon. The consumption of a high fat diet also reduced the failure stress and load of the patellar tendon in both mouse types, and increased Mmp2 expression. ApoE -/- mice exhibited more pronounced reductions in tendon function than wild-type mice, and decreased expression of Col1a1 compared to wild type mice. Human tendon fibroblasts responded to oxLDL by increasing their proliferation and their mRNA levels of MMP2, while decreasing their mRNA levels for COL1A1 and COL3A1. Conclusion The consumption of a high fat diet resulted in deleterious changes in tendon function, and these changes may be explained in part by the effects of oxLDL, which induced a proliferative, matrix-degrading phenotype in human tenocytes. PMID:25502628

  15. Targeting of GFP-Cre to the Mouse Cyp11a1 Locus Both Drives Cre Recombinase Expression in Steroidogenic Cells and Permits Generation of Cyp11a1 Knock Out Mice

    PubMed Central

    O'Hara, Laura; York, Jean Philippe; Zhang, Pumin; Smith, Lee B.

    2014-01-01

    To permit conditional gene targeting of floxed alleles in steroidogenic cell-types we have generated a transgenic mouse line that expresses Cre Recombinase under the regulation of the endogenous Cytochrome P450 side chain cleavage enzyme (Cyp11a1) promoter. Mice Carrying the Cyp11a1-GC (GFP-Cre) allele express Cre Recombinase in fetal adrenal and testis, and adrenal cortex, testicular Leydig cells (and a small proportion of Sertoli cells), theca cells of the ovary, and the hindbrain in postnatal life. Circulating testosterone concentration is unchanged in Cyp11+/GC males, suggesting steroidogenesis is unaffected by loss of one allele of Cyp11a1, mice are grossly normal, and Cre Recombinase functions to recombine floxed alleles of both a YFP reporter gene and the Androgen Receptor (AR) in steroidogenic cells of the testis, ovary, adrenal and hindbrain. Additionally, when bred to homozygosity (Cyp11a1GC/GC), knock-in of GFP-Cre to the endogenous Cyp11a1 locus results in a novel mouse model lacking endogenous Cyp11a1 (P450-SCC) function. This unique dual-purpose model has utility both for those wishing to conditionally target genes within steroidogenic cell types and for studies requiring mice lacking endogenous steroid hormone production. PMID:24404170

  16. Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice.

    PubMed

    Oguro, K; Jover, T; Tanaka, H; Lin, Y; Kojima, T; Oguro, N; Grooms, S Y; Bennett, M V; Zukin, R S

    2001-10-01

    Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

  17. Mice expressing the human CYP7A1 gene in the mouse CYP7A1 knock-out background lack induction of CYP7A1 expression by cholesterol feeding and have increased hypercholesterolemia when fed a high fat diet.

    PubMed

    Chen, Jean Y; Levy-Wilson, Beatriz; Goodart, Sheryl; Cooper, Allen D

    2002-11-08

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the pathway responsible for the formation of the majority of bile acids. Transcription of the gene is regulated by the size of the bile acid pool and dietary and hormonal factors. The farnesoid X receptor and the liver X receptor (LXR) are responsible for regulation by bile acids and cholesterol, respectively. To study the effects of dietary cholesterol and fat upon expression of the human CYP7A1 gene, mice were generated by crossing transgenic mice carrying the human CYP7A1 gene with mice that were homozygous knock-outs (CYP7A1(-/-)). The mice (mCYP7A1(-/-)/hCYP7A1) expressed the human gene at much higher levels than did the transgenics bred in the wild-type background. A diet containing 1% cholic acid reduced the expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice to undetectable levels. Cholestyramine (5%) increased the level of expression of the human gene and the mouse gene. Thus, farnesoid X receptor-mediated regulation was preserved. A diet containing 2% cholesterol increased expression of the mouse gene in wild-type mice, but it did not affect expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice. None of the diets altered the serum cholesterol or triglyceride levels in these mice; 1% cholic acid caused a redistribution of cholesterol from the high density lipoprotein to the low density lipoprotein density in the humanized mice but not in wild-type mice. A diet containing 30% saturated fat and 2% cholesterol caused a decrease in CYP7A1 levels in mCYP7A1(-/-)/hCYP7A1 mice. The serum cholesterol levels rose in all mice fed this diet. The increase was greater in the mCYP7A1(-/-)/hCYP7A1 mice. Together, these data suggest that the lack of an LXR element in the region from -56 to -49 of the human CYP7A1 promoter may account for some of the differences in response to diets between humans and rodents.

  18. The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

    2016-01-01

    The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710. PMID:27746629

  19. TALEN-induced gene knock out in Drosophila.

    PubMed

    Kondo, Takefumi; Sakuma, Tetsushi; Wada, Housei; Akimoto-Kato, Ai; Yamamoto, Takashi; Hayashi, Shigeo

    2014-01-01

    We report here a case study of TALEN-induced gene knock out of the trachealess gene of Drosophila. Two pairs of TALEN constructs caused targeted mutation in the germ line of 39% and 17% of injected animals, respectively. In the extreme case 100% of the progeny of TALEN-injected fly was mutated, suggesting that highly efficient biallelic germ line mutagenesis was achieved. The mutagenic efficiency of the TALEN pairs paralleled their activity of single strand annealing (SSA) assay in cultured cells. All mutations were deletion of 1 to 20 base pairs. Merit and demerit of TALEN-based gene knockout approach compared to other genome editing technologies is discussed. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  20. Characterization of TG2 and TG1-TG2 double knock-out mouse epidermis.

    PubMed

    Pitolli, Consuelo; Pietroni, Valentina; Marekov, Lyuben; Terrinoni, Alessandro; Yamanishi, Kiyofumi; Mazzanti, Cinzia; Melino, Gerry; Candi, Eleonora

    2017-03-01

    Transglutaminases (TGs) are a family of enzymes that catalyse the formation of isopeptide bonds between the γ-carboxamide groups of glutamine residues and the ε-amino groups of lysine residues leading to cross-linking reactions among proteins. Four members, TG1, TG2, TG3, and TG5, of the nine mammalian enzymes are expressed in the skin. TG1, TG3 and TG5 crosslinking properties are fundamental for cornified envelope assembly. In contrast, the role of TG2 in keratinization has never been studied at biochemical level in vivo. In this study, taking advantage of the TG2 knock-out (KO) and TG1 heterozygous mice, we generated and characterized the epidermis of TG1-TG2 double knock-out (DKO) mice. We performed morphological analysis of the epidermis and evaluation of the expression of differentiation markers. In addition, we performed analysis of the amino acid composition from isolated corneocytes. We found a significant change in amino acid composition in TG1KO cornified cell envelopes (CEs) while TG2KO amino acid composition was similar to wild-type CEs. Our results confirm a key role of TG1 in skin differentiation and CE assembly and demonstrate that TG2 is not essential for CE assembly and skin formation.

  1. The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

    PubMed

    Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

    2015-01-01

    Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

  2. Oxygen Knock-Out and Other Studies in -Irradiated Polycrystalline Bi-2212 Superconductor

    NASA Astrophysics Data System (ADS)

    Bandyopadhyay, S. K.; Ghosh, A. K.; Barat, P.; Sen, Pintu; Basu, A. N.; Ghosh, B.

    1997-08-01

    Bulk polycrystalline samples of Bi2Sr2CaCu2O8+x (Bi-2212) have been irradiated with 40 MeV -particles. Tc increases up to a certain dose. The increase in Tc is correlated with the knock-out of oxygen, which has been verified by the determination of the oxygen contents of the irradiated samples by iodometry. A model of the knock-out of oxygen is proposed on the basis of Monte-Carlo TRIM calculations. Resistivity versus temperature of the irradiated samples shows fairly metallic behaviour up to a certain dose. Excess conductivity analysis shows a cross-over from 2D to 3D behaviour in conductivity for the unirradiated sample. However, for irradiated samples, the critical fluctuation regime sets in. The interlayer coupling strengths decrease with the increase in the irradiation dose. The sample with the highest dose shows a nonmetallic behaviour in resistivity. A detailed analysis shows a conductivity behaviour in the nonmetallic region characteristic of three-dimensional variable range hopping of charge carriers.

  3. Norepinephrine transporter knock-out alters expression of the genes connected with antidepressant drugs action.

    PubMed

    Solich, Joanna; Kolasa, Magdalena; Kusmider, Maciej; Faron-Gorecka, Agata; Pabian, Paulina; Zurawek, Dariusz; Szafran-Pilch, Kinga; Dziedzicka-Wasylewska, Marta

    2015-01-12

    Norepinephrine transporter knock-out mice (NET-KO) exhibit depression-resistant phenotypes. They manifest significantly shorter immobility times in both the forced swim test and the tail suspension test. Moreover, biochemical studies have revealed the up-regulation of other monoamine transporters (dopamine and serotonin) in the brains of NET-KO mice, similar to the phenomenon observed after the chronic pharmacological blockade of norepinephrine transporter by desipramine in wild-type (WT) animals. NET-KO mice are also resistant to stress, as we demonstrated previously by measuring plasma corticosterone concentration. In the present study, we used a microdissection technique to separate target brain regions and the TaqMan Low Density Array approach to test the expression of a group of genes in the NET-KO mice compared with WT animals. A group of genes with altered expression were identified in four brain structures (frontal and cingulate cortices, dentate gyrus of hippocampus and basal-lateral amygdala) of NET-KO mice compared with WT mice. These genes are known to be altered by antidepressant drugs administration. The most interesting gene is Crh-bp, which modulates the activity of corticotrophin--releasing hormone (CRH) and several CRH-family members. Generally, genetic disturbances within noradrenergic neurons result in biological changes, such as in signal transduction and intercellular communication, and may be linked to changes in noradrenaline levels in the brains of NET-KO mice.

  4. Zika virus infection of adult and fetal STAT2 knock-out hamsters.

    PubMed

    Siddharthan, Venkatraman; Van Wettere, Arnaud J; Li, Rong; Miao, Jinxin; Wang, Zhongde; Morrey, John D; Julander, Justin G

    2017-07-01

    Zika virus (ZIKV) infection was investigated in adult and fetal STAT2 knock-out (KO) hamsters. Subcutaneous injection of ZIKV of adults resulted in morbidity, mortality, and infection of the uterus, placenta, brain, spinal cord, and testicles, thus providing an opportunity to evaluate congenital ZIKV infection in a second rodent species besides mice. ZIKV-infected cells with morphologies of Sertoli cells and spermatogonia were observed in the testes, which may have implications for sexual transmission and male sterility. Neonates exposed as fetuses to ZIKV at 8 days post-coitus were not smaller than controls. Nevertheless, infectious virus and ZIKV RNA was detected in some, but not all, placentas and fetal brains of KO hamsters. STAT2 KO hamsters may be useful for addressing sexual transmission, pathogenesis, routes of fetal infection, and neurological disease outcomes, and may also be used in antiviral or vaccine studies to identify intervention strategies. Copyright © 2017. Published by Elsevier Inc.

  5. METTL21B is a novel human lysine methyltransferase of translation elongation factor 1A: discovery by CRISPR/Cas9 knock out.

    PubMed

    Hamey, Joshua J; Wienert, Beeke; Quinlan, Kate G R; Wilkins, Marc R

    2017-06-29

    Lysine methylation is widespread on human proteins, however the enzymes that catalyse its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. Here we used the CRISPR/Cas9 system to knock out putative protein methyltransferases METTL21B and METTL23 in K562 cells, to determine if they methylate elongation factor eEF1A. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knock out of METTL21B, and the expected loss of lysine 79 methylation on knock out of EEF1AKMT1. No loss of eEF1A methylation was seen in the METTL23 knock out. Recombinant METTL21B was shown in vitro to catalyse methylation on lysine 165 in eEF1A1 and eEF1A2, confirming it as the methyltransferase responsible for this methylation site. Proteomic analysis by SILAC revealed specific upregulation of large ribosomal subunit proteins in the METTL21B knock out, and changes to further processes related to eEF1A function in knock outs of both METTL21B and EEF1AKMT1. This indicates that the methylation of lysine 165 in human eEF1A has a very specific role. METTL21B exists only in vertebrates, with its target lysine showing similar evolutionary conservation. We suggest METTL21B be renamed eEF1A-KMT3. This is the first study to specifically generate CRISPR/Cas9 knock outs of putative protein methyltransferase genes, for the purpose of substrate discovery and site mapping. Our approach should prove useful for the discovery of further novel methyltransferases, and more generally for the discovery of sites for other protein-modifying enzymes. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  6. Knock-Out Models Reveal New Aquaporin Functions

    PubMed Central

    Verkman, Alan S.

    2013-01-01

    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

  7. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

    PubMed

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-12-15

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

  8. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model

    PubMed Central

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-01-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration. PMID:22983956

  9. Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response

    PubMed Central

    Ojeda-Fernández, Luisa; Recio-Poveda, Lucía; Aristorena, Mikel; Lastres, Pedro; Blanco, Francisco J.; Sanz-Rodríguez, Francisco; Gallardo-Vara, Eunate; de las Casas-Engel, Mateo; Corbí, Ángel; Arthur, Helen M.; Bernabeu, Carmelo; Botella, Luisa M.

    2016-01-01

    Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Engfl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Engfl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients. PMID:27010826

  10. Knock-Outs, Stick-Outs, Cut-Outs: Clipping Paths Separate Objects from Background.

    ERIC Educational Resources Information Center

    Wilson, Bradley

    1998-01-01

    Outlines a six-step process that allows computer operators, using Photoshop software, to create "knock-outs" to precisely define the path that will serve to separate the object from the background. (SR)

  11. Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

    PubMed

    O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

    2014-03-01

    The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

  12. 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). © 2016 The American Society of Photobiology.

  13. Photodynamic therapy and knocking out of single tumor cells by multiphoton excitation processes

    NASA Astrophysics Data System (ADS)

    Riemann, Iris; Fischer, Peter; Koenig, Karsten

    2004-09-01

    Near infrared (NIR) ultrashort laser pulses of 780 nm have been used to induce intracellular photodynamic reactions by nonlinear excitation of porphyrin photosensitizers. Intracellular accumulation and photobleaching of the fluorescent photosensitizers protoporphyrin IX and Photofrin (PF) have been studied by non-resonant two-photon fluorescence excitation of PF and aminolevulinic acid (ALA)-labeled Chinese hamster ovary (CHO) cells. To testify the efficacy of both substrates to induce irreversible destructive effects, the cloning efficiency (CE) of cells exposed to femtosecond pulses of a multiphoton laser scanning microscope (40x/1.3) was determined. In the case of Photofrin accumulation, CEs of 50% and 0% were obtained after 17 laserscans (2 mW?, 16 s/ frame) and 50 scans, respectively. All cells exposed to 50 scans died within 48h after laser exposure. 100 scans were required to induce lethal effects in ALA labeled cells. Sensitizer-free control cells could be scanned 250 times (1.1 h) and more without impact on the reproduction behavior, morphology, and vitality. In addition to the slow phototoxic effect by photooxidation processes, another destructive but immediate effect based on optical breakdown was induced when employing high intense NIR femtosecond laser beams. This was used to optically knock out single tumor cells in living mice (solid Ehrlich-Carcinoma) in a depth of 10 to 100 μm.

  14. Characterization of skeletal muscle in the synemin knock-out mouse

    NASA Astrophysics Data System (ADS)

    García-Pelagio, Karla P.; Muriel, Joaquin; Lovering, Richard M.; Lund, Linda; Bond, Meredith; Bloch, Robert J.

    2014-11-01

    Diseases linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus of skeletal and cardiac muscle and its links to costameres, which connect the sarcomeres to the cell membrane. Synemin is a large IF protein that associates with dystrobrevin, vinculin, and talin at costameres of the cell membrane of striated muscle, as well as with α-actinin and desmin at the Z disks. Synemin can be expressed in either 210 kDa α- or 180 kDa β- alternatively spliced forms. We generated mice null for synemin by homologous recombination to study synemin's function in skeletal muscle. Skeletal muscle in the knock out (syn KO) mouse does not make synemin mRNA or protein. Preliminary characterization of the syn KO mouse suggests that it has a mild skeletal muscle phenotype. The organization of costameres appears to be normal. Treadmill running uphill test results was not significantly affected when compared to controls at any age. More notably, the biomechanical properties of the cell membrane are different in the syn KO, though they are less affected than by the absence of desmin or dystrophin. These results suggest that the viscoelastic properties of the cell membrane-costamere-myofibril complex are significantly influenced by synemin.

  15. CRISPR knock out CTLA-4 enhances the anti-tumor activity of cytotoxic T lymphocytes.

    PubMed

    Shi, Long; Meng, Tongyu; Zhao, Zhilong; Han, Jinsheng; Zhang, Wei; Gao, Fei; Cai, Jianhui

    2017-09-06

    T cell-mediated anti-tumor immunity plays a pivotal role in cancer immune surveillance. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a protein receptor mainly expressed in activated T cells and regulatory T cells. CTLA-4 competes with CD28 for ligand binding and generates inhibitory signals to attenuate T cell activation. The blockade of CTLA-4 mediated immune inhibitory checkpoint has been associated with enhanced anti-tumor immunity. In this study, we use CRISPR-Cas9 system to knock out (KO) CTLA-4 from cytotoxic T lymphocytes (CTLs) and evaluate its effect on the anti-tumor activity of the CTLs. CTLA-4 KO CTLs robustly enhanced tumor cell death by 40% compared to the control and facilitated apoptosis and caspase activities in tumor cells. The knockout of CTLA-4 also increased TNF-α and IFN-γ secretion of the CTLs by approximately 2-fold. The effectiveness of CTLA-4 KO in enhancing anti-tumor activity of the CTLs was verified in vivo using mouse xenograft model. The xenografted mice treated with CTLA-4 KO CTLs demonstrated repressed tumor growth and prolonged survival compared to the control group. Our data suggest that CRISPR targeting CTLA-4 immune checkpoint could significantly improve the anti-tumor activity of CTLs. Copyright © 2017. Published by Elsevier B.V.

  16. Phospholipase D δ knock-out mutants are tolerant to severe drought stress

    PubMed Central

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  17. Phospholipase D δ knock-out mutants are tolerant to severe drought stress.

    PubMed

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; Ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance.

  18. Multiple Cranial Organ Defects after Conditionally Knocking Out Fgf10 in the Neural Crest

    PubMed Central

    Teshima, Tathyane H. N.; Lourenco, Silvia V.; Tucker, Abigail S.

    2016-01-01

    Fgf10 is necessary for the development of a number of organs that fail to develop or are reduced in size in the null mutant. Here we have knocked out Fgf10 specifically in the neural crest driven by Wnt1cre. The Wnt1creFgf10fl/fl mouse phenocopies many of the null mutant defects, including cleft palate, loss of salivary glands, and ocular glands, highlighting the neural crest origin of the Fgf10 expressing mesenchyme surrounding these organs. In contrast tissues such as the limbs and lungs, where Fgf10 is expressed by the surrounding mesoderm, were unaffected, as was the pituitary gland where Fgf10 is expressed by the neuroepithelium. The circumvallate papilla of the tongue formed but was hypoplastic in the conditional and Fgf10 null embryos, suggesting that other sources of FGF can compensate in development of this structure. The tracheal cartilage rings showed normal patterning in the conditional knockout, indicating that the source of Fgf10 for this tissue is mesodermal, which was confirmed using Wnt1cre-dtTom to lineage trace the boundary of the neural crest in this region. The thyroid, thymus, and parathyroid glands surrounding the trachea were present but hypoplastic in the conditional mutant, indicating that a neighboring source of mesodermal Fgf10 might be able to partially compensate for loss of neural crest derived Fgf10. PMID:27826253

  19. Direct calculation of minimal cut sets involving a specific reaction knock-out.

    PubMed

    Tobalina, Luis; Pey, Jon; Planes, Francisco J

    2016-07-01

    The concept of Minimal Cut Sets (MCSs) is used in metabolic network modeling to describe minimal groups of reactions or genes whose simultaneous deletion eliminates the capability of the network to perform a specific task. Previous work showed that MCSs where closely related to Elementary Flux Modes (EFMs) in a particular dual problem, opening up the possibility to use the tools developed for computing EFMs to compute MCSs. Until recently, however, there existed no method to compute an EFM with some specific characteristic, meaning that, in the case of MCSs, the only strategy to obtain them was to enumerate them using, for example, the standard K-shortest EFMs algorithm. In this work, we adapt the recently developed theory to compute EFMs satisfying several constraints to the calculation of MCSs involving a specific reaction knock-out. Importantly, we emphasize that not all the EFMs in the dual problem correspond to real MCSs, and propose a new formulation capable of correctly identifying the MCS wanted. Furthermore, this formulation brings interesting insights about the relationship between the primal and the dual problem of the MCS computation. A Matlab-Cplex implementation of the proposed algorithm is available as a supplementary material fplanes@ceit.es Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. [Construction of genetically engineered strain producing 5- oxomilbemycin by knocking out milF in Steptomyces hygroscopicus HS023].

    PubMed

    Huang, Jun; Lin, Jiatan; Zhou, Min; Bai, Hua

    2015-01-04

    To construct a 5-oxomilbemycin producing strain by knocking out milF gene in Streptomyces hygroscopicus HS023. Plasmid pMSST-ΔmilF was constructed and introduced into milbemycin industrial strain Streptomyces hygroscopicus HS023, and milF mutant F2-18 was selected by PCR amplification. Fermentation experiments showed that no milbemycins was produced in F2-18, but 5-ketomilbemycin, the intermediate of milbemycin, was obviously accumulated, and the fermentation titer was enhanced. Genetically engineered strain can simplify the synthesis of milbemycin oxime and lepimectin chemical from milbemycin.

  1. 'Knock, and it shall be opened': knocking out and knocking in to reveal mechanisms of disease and novel therapies.

    PubMed

    Hacking, Douglas F

    2008-12-01

    Recent significant advances in molecular biology have generated genetically modified bacteria, yeast, nematodes, fruit flies, and fish. However, it is the genetic modification of mammalian model organisms, particularly the mouse, that has the greatest potential to shed light on human development, physiology and pathology in ways that have significant implications for neonatal and paediatric clinical practice. Here, we review some of the techniques for knocking out (inactivating), mutating and knocking in (inserting) selected genes that are important to neonatology and show how this research will lead both to a better understanding of disease and to novel therapies for infants and children.

  2. The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages

    PubMed Central

    Saikolappan, Sankaralingam; Estrella, Jaymie; Sasindran, Smitha J.; Khan, Arshad; Armitige, Lisa Y.; Jagannath, Chinnaswamy; Dhandayuthapani, Subramanian

    2012-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine. PMID:22574140

  3. Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1.

    PubMed

    Li, Li; Zhang, Quanjun; Yang, Huaqiang; Zou, Qingjian; Lai, Chengdan; Jiang, Fei; Zhao, Ping; Luo, Zhiwei; Yang, Jiayin; Chen, Qian; Wang, Yan; Newsome, Philip N; Frampton, Jon; Maxwell, Patrick H; Li, Wenjuan; Chen, Shuhan; Wang, Dongye; Siu, Tak-Shing; Tam, Sidney; Tse, Hung-Fat; Qin, Baoming; Bao, Xichen; Esteban, Miguel A; Lai, Liangxue

    2017-03-17

    Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH(-/-) rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH(-/-) rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH(-/-) rabbits are an attractive alternative for modeling the consequences of HT1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1*

    PubMed Central

    Li, Li; Zhang, Quanjun; Yang, Huaqiang; Zou, Qingjian; Lai, Chengdan; Jiang, Fei; Zhao, Ping; Luo, Zhiwei; Yang, Jiayin; Chen, Qian; Wang, Yan; Newsome, Philip N.; Frampton, Jon; Maxwell, Patrick H.; Li, Wenjuan; Chen, Shuhan; Wang, Dongye; Siu, Tak-Shing; Tam, Sidney; Tse, Hung-Fat; Qin, Baoming; Bao, Xichen; Esteban, Miguel A.; Lai, Liangxue

    2017-01-01

    Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH−/− rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH−/− rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH−/− rabbits are an attractive alternative for modeling the consequences of HT1. PMID:28053091

  5. Mice with Deficient BK Channel Function Show Impaired Prepulse Inhibition and Spatial Learning, but Normal Working and Spatial Reference Memory

    PubMed Central

    Azzopardi, Erin; Ruettiger, Lukas; Ruth, Peter; Schmid, Susanne

    2013-01-01

    Genetic variations in the large-conductance, voltage- and calcium activated potassium channels (BK channels) have been recently implicated in mental retardation, autism and schizophrenia which all come along with severe cognitive impairments. In the present study we investigate the effects of functional BK channel deletion on cognition using a genetic mouse model with a knock-out of the gene for the pore forming α-subunit of the channel. We tested the F1 generation of a hybrid SV129/C57BL6 mouse line in which the slo1 gene was deleted in both parent strains. We first evaluated hearing and motor function to establish the suitability of this model for cognitive testing. Auditory brain stem responses to click stimuli showed no threshold differences between knockout mice and their wild-type littermates. Despite of muscular tremor, reduced grip force, and impaired gait, knockout mice exhibited normal locomotion. These findings allowed for testing of sensorimotor gating using the acoustic startle reflex, as well as of working memory, spatial learning and memory in the Y-maze and the Morris water maze, respectively. Prepulse inhibition on the first day of testing was normal, but the knockout mice did not improve over the days of testing as their wild-type littermates did. Spontaneous alternation in the y-maze was normal as well, suggesting that the BK channel knock-out does not impair working memory. In the Morris water maze knock-out mice showed significantly slower acquisition of the task, but normal memory once the task was learned. Thus, we propose a crucial role of the BK channels in learning, but not in memory storage or recollection. PMID:24303038

  6. The effect of neuronal conditional knock-out of peroxisome proliferator-activated receptors in the MPTP mouse model of Parkinson's disease.

    PubMed

    Mounsey, R B; Martin, H L; Nelson, M C; Evans, R M; Teismann, P

    2015-08-06

    Activation of peroxisome proliferator-activated receptors (PPARs), namely PPARγ and PPARδ, has been shown to provide neuroprotection in a number of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease (PD). The observed neuroprotective effects in experimental models of PD have been linked to anti-oxidant and anti-inflammatory actions. This study aimed to analyze the full influence of these receptors in neuroprotection by generating a nerve cell-specific conditional knock-out of these receptors and subjecting these genetically modified mice to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to model dopaminergic degeneration. Mice null for both receptors show the lowest levels of tyrosine hydroxylase (TH)-positive cell bodies following MPTP administration. Presence of one or both these receptors show a trend toward protection against this degeneration, as higher dopaminergic cell immunoreactivity and striatal monoamine levels are evident. These data supplement recent studies that have elected to use agonists of the receptors to regulate immune responses. The results place further importance on the activation of PPARs and the neuroprotective roles these have in inflammatory processes linked to neurodegenerative processes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. The effect of neuronal conditional knock-out of peroxisome proliferator-activated receptors in the MPTP mouse model of Parkinson’s disease

    PubMed Central

    Mounsey, R.B.; Martin, H.L.; Nelson, M.C.; Evans, R.M.; Teismann, P.

    2015-01-01

    Activation of peroxisome proliferator-activated receptors (PPARs), namely PPARγ and PPARδ, has been shown to provide neuroprotection in a number of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (PD). The observed neuroprotective effects in experimental models of PD have been linked to anti-oxidant and anti-inflammatory actions. This study aimed to analyze the full influence of these receptors in neuroprotection by generating a nerve cell-specific conditional knock-out of these receptors and subjecting these genetically modified mice to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to model dopaminergic degeneration. Mice null for both receptors show the lowest levels of tyrosine hydroxylase (TH)-positive cell bodies following MPTP administration. Presence of one or both these receptors show a trend toward protection against this degeneration, as higher dopaminergic cell immunoreactivity and striatal monoamine levels are evident. These data supplement recent studies that have elected to use agonists of the receptors to regulate immune responses. The results place further importance on the activation of PPARs and the neuroprotective roles these have in inflammatory processes linked to neurodegenerative processes. PMID:26028469

  8. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    PubMed Central

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells. PMID:26436575

  9. Tactile Defensiveness and Impaired Adaptation of Neuronal Activity in the Fmr1 Knock-Out Mouse Model of Autism.

    PubMed

    He, Cynthia X; Cantu, Daniel A; Mantri, Shilpa S; Zeiger, William A; Goel, Anubhuti; Portera-Cailliau, Carlos

    2017-07-05

    Sensory hypersensitivity is a common symptom in autism spectrum disorders (ASDs), including fragile X syndrome (FXS), and frequently leads to tactile defensiveness. In mouse models of ASDs, there is mounting evidence of neuronal and circuit hyperexcitability in several brain regions, which could contribute to sensory hypersensitivity. However, it is not yet known whether or how sensory stimulation might trigger abnormal sensory processing at the circuit level or abnormal behavioral responses in ASD mouse models, especially during an early developmental time when experience-dependent plasticity shapes such circuits. Using a novel assay, we discovered exaggerated motor responses to whisker stimulation in young Fmr1 knock-out (KO) mice (postnatal days 14-16), a model of FXS. Adult Fmr1 KO mice actively avoided a stimulus that was innocuous to wild-type controls, a sign of tactile defensiveness. Using in vivo two-photon calcium imaging of layer 2/3 barrel cortex neurons expressing GCaMP6s, we found no differences between wild-type and Fmr1 KO mice in overall whisker-evoked activity, though 45% fewer neurons in young Fmr1 KO mice responded in a time-locked manner. Notably, we identified a pronounced deficit in neuronal adaptation to repetitive whisker stimulation in both young and adult Fmr1 KO mice. Thus, impaired adaptation in cortical sensory circuits is a potential cause of tactile defensiveness in autism.SIGNIFICANCE STATEMENT We use a novel paradigm of repetitive whisker stimulation and in vivo calcium imaging to assess tactile defensiveness and barrel cortex activity in young and adult Fmr1 knock-out mice, the mouse model of fragile X syndrome (FXS). We describe evidence of tactile defensiveness, as well as a lack of L2/3 neuronal adaptation in barrel cortex, during whisker stimulation. We propose that a defect in sensory adaptation within local neuronal networks, beginning at a young age and continuing into adulthood, likely contributes to sensory overreactivity

  10. Orexin/hypocretin and histamine: distinct roles in the control of wakefulness demonstrated using knock-out mouse models.

    PubMed

    Anaclet, Christelle; Parmentier, Régis; Ouk, Koliane; Guidon, Gérard; Buda, Colette; Sastre, Jean-Pierre; Akaoka, Hidéo; Sergeeva, Olga A; Yanagisawa, Masashi; Ohtsu, Hiroshi; Franco, Patricia; Haas, Helmut L; Lin, Jian-Sheng

    2009-11-18

    To determine the respective role played by orexin/hypocretin and histamine (HA) neurons in maintaining wakefulness (W), we characterized the behavioral and sleep-wake phenotypes of orexin (Ox) knock-out (-/-) mice and compared them with those of histidine-decarboxylase (HDC, HA-synthesizing enzyme)-/- mice. While both mouse strains displayed sleep fragmentation and increased paradoxical sleep (PS), they presented a number of marked differences: (1) the PS increase in HDC(-/-) mice was seen during lightness, whereas that in Ox(-/-) mice occurred during darkness; (2) contrary to HDC(-/-), Ox(-/-) mice had no W deficiency around lights-off, nor an abnormal EEG and responded to a new environment with increased W; (3) only Ox(-/-), but not HDC(-/-) mice, displayed narcolepsy and deficient W when faced with motor challenge. Thus, when placed on a wheel, wild-type (WT), but not littermate Ox(-/-) mice, voluntarily spent their time in turning it and as a result, remained highly awake; this was accompanied by dense c-fos expression in many areas of their brains, including Ox neurons in the dorsolateral hypothalamus. The W and motor deficiency of Ox(-/-) mice was due to the absence of Ox because intraventricular dosing of orexin-A restored their W amount and motor performance whereas SB-334867 (Ox1-receptor antagonist, i.p.) impaired W and locomotion of WT mice during the test. These data indicate that Ox, but not HA, promotes W through enhanced locomotion and suggest that HA and Ox neurons exert a distinct, but complementary and synergistic control of W: the neuropeptide being more involved in its behavioral aspects, whereas the amine is mainly responsible for its qualitative cognitive aspects and cortical EEG activation.

  11. Knock-out drugs: their prevalence, modes of action, and means of detection.

    PubMed

    Madea, Burkhard; Musshoff, Frank

    2009-05-01

    Knock-out drugs are used to facilitate the commission of a crime, generally either robbery or sexual assault. Although media reports on the use of knock-out drugs have become more frequent, there are no robust epidemiological data on the incidence of drug-facilitated robbery or sexual assault, presumably because many crimes of these types do not enter into official statistics. The authors describe the modes of action and toxicological means of detection of the substances most frequently used as knock-out drugs on the basis of a selective literature research on the terms "drug-facilitated sexual assaults" (DFSA) and "drug-facilitated crimes" (DFC). The most frequently used drug in cases of sexual assault is still alcohol (ca. 40% to 60%), followed by illegal drugs (cannabis, cocaine). The presence of involuntarily consumed medications and drugs of abuse is demonstrated by routine toxicological analysis only in relatively few cases (ca. 2%). The substances most commonly found are benzodiazepines, followed by other hypnotics. In Europe, the illegal substance gamma-hydroxybutyric acid (GHB, "Liquid Ecstasy"), often mentioned as a "date-rape drug," is only rarely detected with sufficient medicolegal certainty. This may be due to its rapid elimination (it is detectable in blood for up to 8 hours, in urine for up to 12 hours) as well as its physiological occurrence in the body. If the toxicological analysis of blood and urine is negative in a case of suspected DFSA, then the analysis of a hair sample about four weeks after the assault can detect the presence of drugs consumed at that time. If the victim has long hair, it may be possible to detect knock-out drugs taken more than four weeks earlier. In Europe, convictions for drug-facilitated crimes are comparatively rare, mainly because of the difficulty of demonstrating conclusive evidence. A careful medical history and physical examination and the careful taking of biological samples for toxicological analysis form the

  12. Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies.

    PubMed

    Lin, F; Fukuoka, Y; Spicer, A; Ohta, R; Okada, N; Harris, C L; Emancipator, S N; Medof, M E

    2001-10-01

    Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.

  13. A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Taurine Distribution in Various Tissues With and Without Taurine Supplementation.

    PubMed

    Park, Eunkyue; Park, Seung Yong; Cho, In Soo; Kim, Bo Sook; Schuller-Levis, Georgia

    2017-01-01

    Taurine, a sulfur containing amino acid, has various physiological functions including development of the eye and brain, immune function, reproduction, osmo-regulatory function as well as anti-oxidant and anti-inflammatory activities. In order to understand the physiological role, we developed taurine deficient mice deleting a rate-liming enzyme, cysteine sulfinic acid decarboxylase (CSAD) for biosynthesis of taurine. Taurine was measured in various tissues including the liver, brain, lung, spleen, thymus, pancreas, heart, muscle and kidney as well as plasma from CSAD knock-out mice (CSAD KO) with and without treatment of taurine in the drinking water at the age of 2 months (2 M). Taurine was determined using HPLC as a phenylisothiocyanate derivative of taurine at 254 nm. Taurine concentrations in the liver and kidney from homozygotes of CSAD KO (HO), in which CSAD level is high, were 90% and 70% lower than WT, respectively. Taurine concentrations in the brain, spleen and lung, where CSAD level is low, were 21%, 20% and 28% lower than WT, respectively. At 2 M, 1% taurine treatment of HO restored taurine concentrations in all tissues compared to that of WT. To select an appropriate taurine treatment, HO were treated with various concentrations (0.05, 0.2, 1%) of taurine for 4 months (4 M). Restoration of taurine in all tissues except the liver, kidney and lung requires 0.05% taurine to be restored to that of WT. The liver and kidney restore taurine back to WT with 0.2% taurine. To examine which enzymes influence taurine concentrations in various tissues from WT and HO at 2 M, expression of five taurine-related enzymes, two antioxidant enzymes as well as lactoferrin (Lft) and prolactin receptor (Prlr) was determined using RT(2) qPCR. The expression of taurine transporter in the liver, brain, muscle and kidney from HO was increased except in the lung. Our data showed expression of glutamate decarboxylase-like 1(Gadl-1) was increased in the brain and muscle in HO

  14. Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

    PubMed

    Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.

  15. Monitoring concussion in a knocked-out boxer by CSF biomarker analysis.

    PubMed

    Neselius, Sanna; Brisby, Helena; Granholm, Fredrik; Zetterberg, Henrik; Blennow, Kaj

    2015-09-01

    Concussion is common in many sports, and the incidence is increasing. The medical consequences after a sport-related concussion have received increased attention in recent years since it is known that concussions cause axonal and glial damage, which disturbs the cerebral physiology and makes the brain more vulnerable for additional concussions. This study reports on a knocked-out amateur boxer in whom cerebrospinal fluid (CSF) neurofilament light (NFL) protein, reflecting axonal damage, was used to identify and monitor brain damage. CSF NFL was markedly increased during 36 weeks, suggesting that neuronal injury persists longer than expected after a concussion. CSF biomarker analysis may be valuable in the medical counselling of concussed athletes and in return-to-play considerations.

  16. Transgenic gene knock-outs: functional genomics and therapeutic target selection.

    PubMed

    Harris, S; Foord, S M

    2000-11-01

    The completion of the first draft of the human genome presents both a tremendous opportunity and enormous challenge to the pharmaceutical industry since the whole community, with few exceptions, will soon have access to the same pool of candidate gene sequences from which to select future therapeutic targets. The commercial imperative to select and pursue therapeutically relevant genes from within the overall content of the genome will be particularly intense for those gene families that currently represent the chemically tractable or 'drugable' gene targets. As a consequence the emphasis within exploratory research has shifted towards the evaluation and adoption of technology platforms that can add additional value to the gene selection process, either through functional studies or direct/indirect measures of disease alignment e.g., genetics, differential gene expression, proteomics, tissue distribution, comparative species data etc. The selection of biological targets for the development of potential new medicines relies, in part, on the quality of the in vivo biological data that correlates a particular molecular target with the underlying pathophysiology of a disease. Within the pharmaceutical industry, studies employing transgenic animals and, in particular, animals with specific gene deletions are playing an increasingly important role in the therapeutic target gene selection, drug candidate selection and product development phases of the overall drug discovery process. The potential of phenotypic information from gene knock-outs to contribute to a high-throughput target selection/validation strategy has hitherto been limited by the resources required to rapidly generate and characterise a large number of knock-out transgenics in a timely fashion. The offerings of several companies that provide an opportunity to overcome these hurdles, albeit at a cost, are assessed with respect to the strategic business needs of the pharmaceutical industry.

  17. Age-Dependent Deficits in Fear Learning in Heterozygous BDNF Knock-Out Mice

    ERIC Educational Resources Information Center

    Endres, Thomas; Lessmann, Volkmar

    2012-01-01

    Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF…

  18. Subregion-Specific p300 Conditional Knock-Out Mice Exhibit Long-Term Memory Impairments

    ERIC Educational Resources Information Center

    Oliveira, Ana M. M.; Estevez, Marcel A.; Hawk, Joshua D.; Grimes, Shannon; Brindle, Paul K.; Abel, Ted

    2011-01-01

    Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting…

  19. Age-Dependent Deficits in Fear Learning in Heterozygous BDNF Knock-Out Mice

    ERIC Educational Resources Information Center

    Endres, Thomas; Lessmann, Volkmar

    2012-01-01

    Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF…

  20. Subregion-Specific p300 Conditional Knock-Out Mice Exhibit Long-Term Memory Impairments

    ERIC Educational Resources Information Center

    Oliveira, Ana M. M.; Estevez, Marcel A.; Hawk, Joshua D.; Grimes, Shannon; Brindle, Paul K.; Abel, Ted

    2011-01-01

    Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting…

  1. Ammonia excretion in Caenorhabditis elegans: Physiological and molecular characterization of the rhr-2 knock-out mutant.

    PubMed

    Adlimoghaddam, Aida; O'Donnell, Michael J; Kormish, Jay; Banh, Sheena; Treberg, Jason R; Merz, David; Weihrauch, Dirk

    2016-05-01

    Previous studies have shown the free living soil nematode Caenorhabditis elegans (N2 strain) to be ammonotelic. Ammonia excretion was suggested to take place partially via the hypodermis, involving the Na(+)/K(+)-ATPase (NKA), V-ATPase (VAT), carbonic anhydrase, NHX-3 and a functional microtubule network and at least one Rh-like ammonia transporter RHR-1. In the current study, we show that a second Rh-protein, RHR-2, is highly expressed in the hypodermis, here also in the apical membrane of that tissue. To further characterize the role of RHR-2 in ammonia excretion, a knock-out mutant rhr-2 (ok403), further referred to as ∆rhr-2, was employed. Compared to wild-type worms (N2), this mutant showed a lower rate of ammonia excretion and a lower hypodermal H(+) excretion rate. At the same time rhr-1, nka, vat, and nhx-3 showed higher mRNA expression levels when compared to N2. Also, in contrast to N2 worms, ∆rhr-2 did not show enhanced ammonia excretion rates when exposed to a low pH environment, suggesting that RHR-2 represents the apical NH3 pathway that allows ammonia trapping via the hypodermis in N2 worms. A hypothetical model for the mechanism of hypodermal ammonia excretion is proposed on the basis of data in this and previous investigations. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Knocking out P2X receptors reduces transmitter secretion in taste buds

    PubMed Central

    Huang, Yijen A.; Stone, Leslie M.; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C.; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E.; Kinnamon, Sue C.; Roper, Stephen D.

    2011-01-01

    In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors (P2X2 and P2X3 double knockout, or “DKO” mice). The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca2+ in taste Receptor (Type II) cells from DKO mice, as from wild type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we employed reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion. PMID:21940456

  3. Enhancement in colonization of bovine spermatogonial stem cells following addition of knock-out serum replacement to culture medium

    PubMed Central

    Youssefi, Reza; Tajik, Parviz; Movahedin, Mansoureh; Akbarinejad, Vahid

    2016-01-01

    Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Knock-out serum replacement (KSR) has been reported to enhance the proliferation of murine SSCs and human embryonic stem cells. The aim of the present study was to investigate the effect of KSR versus fetal bovine serum (FBS) and their interaction on colonization of bovine SSCs in vitro. When FBS (10%) was replaced with KSR (10%), a significant increase in the colonization of SSCs and the expression of Thy1, as marker for enrichment of SSCs, was observed. It was revealed that the lesser proliferative effect of FBS as well as the greater proliferative impact of KSR on SSCs colonization were not irreversible as cells having been cultured with FBS (10%) for three days with low colonization showed high rate of colonization in response to KSR (10%) and cells having been cultured with KSR (10%) with high colonization experienced low rate of colonization in response to FBS (10%). Further, it was shown that FBS did not contain factors inhibiting SSCs colonization and it simply lacked factors essential for SSCs proliferation because the combination of FBS (5%) and KSR (5%) resulted in even greater rate of colonization than did KSR (10%). In conclusion, the present study showed that addition of KSR to culture medium would significantly increase SSCs proliferation. PMID:28144417

  4. Enhancement in colonization of bovine spermatogonial stem cells following addition of knock-out serum replacement to culture medium.

    PubMed

    Youssefi, Reza; Tajik, Parviz; Movahedin, Mansoureh; Akbarinejad, Vahid

    2016-01-01

    Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Knock-out serum replacement (KSR) has been reported to enhance the proliferation of murine SSCs and human embryonic stem cells. The aim of the present study was to investigate the effect of KSR versus fetal bovine serum (FBS) and their interaction on colonization of bovine SSCs in vitro. When FBS (10%) was replaced with KSR (10%), a significant increase in the colonization of SSCs and the expression of Thy1, as marker for enrichment of SSCs, was observed. It was revealed that the lesser proliferative effect of FBS as well as the greater proliferative impact of KSR on SSCs colonization were not irreversible as cells having been cultured with FBS (10%) for three days with low colonization showed high rate of colonization in response to KSR (10%) and cells having been cultured with KSR (10%) with high colonization experienced low rate of colonization in response to FBS (10%). Further, it was shown that FBS did not contain factors inhibiting SSCs colonization and it simply lacked factors essential for SSCs proliferation because the combination of FBS (5%) and KSR (5%) resulted in even greater rate of colonization than did KSR (10%). In conclusion, the present study showed that addition of KSR to culture medium would significantly increase SSCs proliferation.

  5. Arterial Remodeling in B-Type Natriuretic Peptide Knock-Out Females

    PubMed Central

    Holditch, Sara J.; Schreiber, Claire A.; Burnett, John C.; Ikeda, Yasuhiro

    2016-01-01

    Sexual dimorphisms are recognized in cardiovascular conditions such as hypertension, stroke, thrombosis and vasculitis. B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. The anti-hypertensive, vasodilatory, anti-fibrotic, and anti-hypertrophic properties of BNP are well established in male animal models. Although circulating BNP levels are higher in women, when compared to age-matched men, the cardiovascular protective propensity of BNP in females is poorly understood. We assessed the cardiovascular consequences of BNP deletion in genetically null (Nppb−/−) female rat lines. Throughout the study, blood pressure (BP) remained uninfluenced by genotype, and cardiorenal consequences of BNP knock out remained minor. Unexpectedly, approximately 60% of Nppb−/− females developed mesenteric polyarteritis-nodosa (PAN)-like vasculitis in their life span, some as early as 4 months of age. Mesenteric lesions involved intense arterial remodeling, progressive inflammation, occluded lumens, and less frequently intestinal necrosis and multiple visceral arterial aneurysms. Cumulative pathologies resulted in a significant decline in survival of the Nppb−/− female. This study highlights BNP’s vasoprotective propensity, bringing to light a possible sex specific difference in the cardiovascular protection provided by BNP. Defects in the BNP/GC-A/cGMP pathway may play a role in arteriopathies in women, while GC-A agonists may provide effective therapy for arteritis. PMID:27162120

  6. Granulin Knock Out Zebrafish Lack Frontotemporal Lobar Degeneration and Neuronal Ceroid Lipofuscinosis Pathology

    PubMed Central

    Solchenberger, Barbara; Russell, Claire; Kremmer, Elisabeth; Haass, Christian; Schmid, Bettina

    2015-01-01

    Loss of function mutations in granulin (GRN) are linked to two distinct neurological disorders, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). It is so far unknown how a complete loss of GRN in NCL and partial loss of GRN in FTLD can result in such distinct diseases. In zebrafish, there are two GRN homologues, Granulin A (Grna) and Granulin B (Grnb). We have generated stable Grna and Grnb loss of function zebrafish mutants by zinc finger nuclease mediated genome editing. Surprisingly, the grna and grnb single and double mutants display neither spinal motor neuron axonopathies nor a reduced number of myogenic progenitor cells as previously reported for Grna and Grnb knock down embryos. Additionally, grna−/−;grnb−/− double mutants have no obvious FTLD- and NCL-related biochemical and neuropathological phenotypes. Taken together, the Grna and Grnb single and double knock out zebrafish lack any obvious morphological, pathological and biochemical phenotypes. Loss of zebrafish Grna and Grnb might therefore either be fully compensated or only become symptomatic upon additional challenge. PMID:25785851

  7. Mice lacking hippocampal left-right asymmetry show non-spatial learning deficits.

    PubMed

    Shimbo, Akihiro; Kosaki, Yutaka; Ito, Isao; Watanabe, Shigeru

    2017-08-31

    Left-right asymmetry is known to exist at several anatomical levels in the brain and recent studies have provided further evidence to show that it also exists at a molecular level in the hippocampal CA3-CA1 circuit. The distribution of N-methyl-d-aspartate (NMDA) receptor NR2B subunits in the apical and basal synapses of CA1 pyramidal neurons is asymmetrical if the input arrives from the left or right CA3 pyramidal neurons. In the present study, we examined the role of hippocampal asymmetry in cognitive function using β2-microglobulin knock-out (β2m KO) mice, which lack hippocampal asymmetry. We tested β2m KO mice in a series of spatial and non-spatial learning tasks and compared the performances of β2m KO and C57BL6/J wild-type (WT) mice. The β2m KO mice appeared normal in both spatial reference memory and spatial working memory tasks but they took more time than WT mice in learning the two non-spatial learning tasks (i.e., a differential reinforcement of lower rates of behavior (DRL) task and a straight runway task). The β2m KO mice also showed less precision in their response timing in the DRL task and showed weaker spontaneous recovery during extinction in the straight runway task. These results indicate that hippocampal asymmetry is important for certain characteristics of non-spatial learning. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

    PubMed

    Meek, Stephen; Thomson, Alison J; Sutherland, Linda; Sharp, Matthew G F; Thomson, Julie; Bishop, Valerie; Meddle, Simone L; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K; Gill, Andrew C; Burdon, Tom

    2016-05-17

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.

  9. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease

    PubMed Central

    Meek, Stephen; Thomson, Alison J.; Sutherland, Linda; Sharp, Matthew G. F.; Thomson, Julie; Bishop, Valerie; Meddle, Simone L.; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K.; Gill, Andrew C.; Burdon, Tom

    2016-01-01

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour. PMID:27185277

  10. Relevant feature set estimation with a knock-out strategy and random forests.

    PubMed

    Ganz, Melanie; Greve, Douglas N; Fischl, Bruce; Konukoglu, Ender

    2015-11-15

    Group analysis of neuroimaging data is a vital tool for identifying anatomical and functional variations related to diseases as well as normal biological processes. The analyses are often performed on a large number of highly correlated measurements using a relatively smaller number of samples. Despite the correlation structure, the most widely used approach is to analyze the data using univariate methods followed by post-hoc corrections that try to account for the data's multivariate nature. Although widely used, this approach may fail to recover from the adverse effects of the initial analysis when local effects are not strong. Multivariate pattern analysis (MVPA) is a powerful alternative to the univariate approach for identifying relevant variations. Jointly analyzing all the measures, MVPA techniques can detect global effects even when individual local effects are too weak to detect with univariate analysis. Current approaches are successful in identifying variations that yield highly predictive and compact models. However, they suffer from lessened sensitivity and instabilities in identification of relevant variations. Furthermore, current methods' user-defined parameters are often unintuitive and difficult to determine. In this article, we propose a novel MVPA method for group analysis of high-dimensional data that overcomes the drawbacks of the current techniques. Our approach explicitly aims to identify all relevant variations using a "knock-out" strategy and the Random Forest algorithm. In evaluations with synthetic datasets the proposed method achieved substantially higher sensitivity and accuracy than the state-of-the-art MVPA methods, and outperformed the univariate approach when the effect size is low. In experiments with real datasets the proposed method identified regions beyond the univariate approach, while other MVPA methods failed to replicate the univariate results. More importantly, in a reproducibility study with the well-known ADNI dataset

  11. Evaluation of cimi-shield knock-out bed bug eliminator against house fly (Musca domestica) adults.

    USDA-ARS?s Scientific Manuscript database

    Cimi-Shield Knock-Out (CSKO) Bed Bug Eliminator is a green treatment labeled for use against bed bugs, carpet beetles, ants, roaches, fleas, ticks, silverfish, millipedes and centipedes. The active ingredient is soybean oil. If CSKO is formulated according to label instructions and sprayed directly ...

  12. Evaluation of cimi-shield knock-out bed bug eliminator against house fly (Musca domestica) adults

    USDA-ARS?s Scientific Manuscript database

    Cimi-Shield Knock-Out (CSKO) Bed Bug Eliminator is a green treatment labeled for use against bed bugs, carpet beetles, ants, roaches, fleas, ticks, silverfish, millipedes and centipedes. The active ingredient is soybean oil. If CSKO is formulated according to label instructions and sprayed directly ...

  13. NCAM-deficient mice show prominent abnormalities in serotonergic and BDNF systems in brain - Restoration by chronic amitriptyline.

    PubMed

    Aonurm-Helm, Anu; Anier, Kaili; Zharkovsky, Tamara; Castrén, Eero; Rantamäki, Tomi; Stepanov, Vladimir; Järv, Jaak; Zharkovsky, Alexander

    2015-12-01

    Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice.

  14. Comparative proteomic analysis of silkworm fat body after knocking out fibroin heavy chain gene: a novel insight into cross-talk between tissues.

    PubMed

    Chen, Quanmei; Ma, Zhengang; Wang, Xin; Li, Zhiqing; Zhang, Yan; Ma, Sanyuan; Zhao, Ping; Xia, Qingyou

    2015-09-01

    Cross-talk between tissues plays key roles in development of organisms; however, there are few researches on cross-talk between tissues in insects. Our previous studies showed that the pupal body weight was elevated after knocking out the fibroin heavy chain gene (BmFib-H), whereas the gene specifically expressed in silk glands of silkworm. Hence, the mutant is a good material for studying the cross-talk between tissues. It is considered that the fat body of silkworm during larval stage is used to store nutrients for pupal development. Herein, comparative proteomic of fat body on the 5th day of fifth instar was performed between BmFib-H gene knock-out Bombyx mori line (FGKO) and its wide-type Dazao. These results revealed that a single gene knock-out in silk gland triggered large-scale metabolic pathways changes in fat body. The levels of proteins involved in glycolysis/gluconeogenesis, pentose phosphate pathway, and glycine-serine biosynthetic pathway were down-regulated in the FGKO fat body. In contrast, the abundances of many proteins participating in protein synthesis, including ribosomal proteins, eukaryotic translation initiation factor, and elongation factor, were up-regulated. Moreover, the concentrations of glycogen and proteins in the FGKO fat body were greatly increased. These findings provided a novel insight into the cross-talk between silk gland and fat body in silkworm, and the presence of cross-talk between silk gland and fat body could regulate the redistribution of nutrients in the FGKO fat body leading to the increase of the pupal weight.

  15. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

    PubMed Central

    Toscano, Miguel G.; Anderson, Per; Muñoz, Pilar; Lucena, Gema; Cobo, Marién; Benabdellah, Karim; Gregory, Philip D.; Holmes, Michael C.; Martin, Francisco

    2013-01-01

    SUMMARY Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed

  16. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome.

    PubMed

    Toscano, Miguel G; Anderson, Per; Muñoz, Pilar; Lucena, Gema; Cobo, Marién; Benabdellah, Karim; Gregory, Philip D; Holmes, Michael C; Martin, Francisco

    2013-03-01

    Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

  17. Hearts of surviving MLP-KO mice show transient changes of intracellular calcium handling.

    PubMed

    Kemecsei, Péter; Miklós, Zsuzsanna; Bíró, Tamás; Marincsák, Rita; Tóth, Balázs I; Komlódi-Pásztor, Edina; Barnucz, Eniko; Mirk, Eva; Van der Vusse, Ger J; Ligeti, László; Ivanics, Tamás

    2010-09-01

    The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca(i)(2+)) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca(i)(2+) cycling, myocardial Ca(i)(2+)-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with beta-agonist isoproterenol and the sarcoplasmic reticular Ca(2+)-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca(2+) handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. beta-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca(i)(2+)-level and a decrease in Ca(2+)-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca(i)(2+) handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.

  18. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

    PubMed

    Crispo, M; Mulet, A P; Tesson, L; Barrera, N; Cuadro, F; dos Santos-Neto, P C; Nguyen, T H; Crénéguy, A; Brusselle, L; Anegón, I; Menchaca, A

    2015-01-01

    While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

  19. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

    PubMed Central

    Crispo, M.; Mulet, A. P.; Tesson, L.; Barrera, N.; Cuadro, F.; dos Santos-Neto, P. C.; Nguyen, T. H.; Crénéguy, A.; Brusselle, L.; Anegón, I.; Menchaca, A.

    2015-01-01

    While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

  20. Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

    PubMed Central

    Ando, Kunie; Leroy, Karelle; Héraud, Céline; Yilmaz, Zehra; Authelet, Michèle; Suain, Valèrie; De Decker, Robert; Brion, Jean-Pierre

    2011-01-01

    Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau−/− mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau−/− mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies. PMID:21281813

  1. Life-long norepinephrine transporter (NET) knock-out leads to the increase in the NET mRNA in brain regions rich in norepinephrine terminals.

    PubMed

    Solich, Joanna; Kolasa, Magdalena; Kusmider, Maciej; Pabian, Paulina; Faron-Gorecka, Agata; Zurawek, Dariusz; Szafran-Pilch, Kinga; Kedracka-Krok, Sylwia; Jankowska, Urszula; Swiderska, Bianka; Dziedzicka-Wasylewska, Marta

    2015-08-01

    These studies aimed to identify the genes differentially expressed in the frontal cortex of mice bearing a life-long norepinephrine transporter knock-out (NET-KO) and wild-type animals (WT). Differences in gene expression in the mouse frontal cortex were studied using a whole-genome microarray approach. Using an alternative approach, i.e. RT-PCR (reverse transcription polymerase chain reaction) with primers complementary to various exons of the NET gene, as well as TaqMan arrays, the level of mRNA encoding the NET in other brain regions of the NET-KO mice was also examined. The analyses revealed a group of 92 transcripts (27 genes) that differentiated the NET-KO mice from the WT mice. Surprisingly, the studies have shown that the mRNA encoding NET accumulated in the brain regions rich in norepinephrine nerve endings in the NET-KO mice. Because there is no other source of NET mRNA besides the noradrenergic terminals in the brain regions studied, these results might speak in favor of the presence of mRNA in axon terminals. RNA-Binding Protein Immunoprecipitation approach indicated that mRNA encoding NET was detected in the Ago2 protein/mRNA complex. In addition, the amount of Ago2 protein in the frontal cortex was significantly higher in NET-KO mice as compared with that of the WT animals. These results are important for further characterization of the NET-KO mice, which - besides other merits - might serve as a good model to study the fate of truncated mRNA in neurons.

  2. Taurine depletion caused by knocking out the taurine transporter gene leads to cardiomyopathy with cardiac atrophy.

    PubMed

    Ito, Takashi; Kimura, Yasushi; Uozumi, Yoriko; Takai, Mika; Muraoka, Satoko; Matsuda, Takahisa; Ueki, Kei; Yoshiyama, Minoru; Ikawa, Masahito; Okabe, Masaru; Schaffer, Stephen W; Fujio, Yasushi; Azuma, Junichi

    2008-05-01

    The sulfur-containing beta-amino acid, taurine, is the most abundant free amino acid in cardiac and skeletal muscle. Although its physiological function has not been established, it is thought to play an important role in ion movement, calcium handling, osmoregulation and cytoprotection. To begin examining the physiological function of taurine, we generated taurine transporter- (TauT-) knockout mice (TauTKO), which exhibited a deficiency in myocardial and skeletal muscle taurine content compared with their wild-type littermates. The TauTKO heart underwent ventricular remodeling, characterized by reductions in ventricular wall thickness and cardiac atrophy accompanied with the smaller cardiomyocytes. Associated with the structural changes in the heart was a reduction in cardiac output and increased expression of heart cardiac failure (fetal) marker genes, such as ANP, BNP and beta-MHC. Moreover, ultrastructural damage to the myofilaments and mitochondria was observed. Further, the skeletal muscle of the TauTKO mice also exhibited decreased cell volume, structural defects and a reduction of exercise endurance capacity. Importantly, the expression of Hsp70, ATA2 and S100A4, which are upregulated by osmotic stress, was elevated in both heart and skeletal muscle of the TauTKO mice. Taurine depletion causes cardiomyocyte atrophy, mitochondrial and myofiber damage and cardiac dysfunction, effects likely related to the actions of taurine. Our data suggest that multiple actions of taurine, including osmoregulation, regulation of mitochondrial protein expression and inhibition of apoptosis, collectively ensure proper maintenance of cardiac and skeletal muscular structure and function.

  3. Knock out of the PHOSPHATE 2 Gene TaPHO2-A1 Improves Phosphorus Uptake and Grain Yield under Low Phosphorus Conditions in Common Wheat

    PubMed Central

    Ouyang, Xiang; Hong, Xia; Zhao, Xueqiang; Zhang, Wei; He, Xue; Ma, Wenying; Teng, Wan; Tong, Yiping

    2016-01-01

    MiR399 and its target PHOSPHATE2 (PHO2) play pivotal roles in phosphate signaling in plants. Loss of function mutation in PHO2 leads to excessive Pi accumulation in shoots and growth retardation in diploid plants like Arabidopsis thaliana and rice (Oryza sativa). Here we isolated three PHO2 homologous genes TaPHO2-A1, -B1 and -D1 from hexaploid wheat (Triticum aestivum). These TaPHO2 genes all contained miR399-binding sites and were able to be degraded by tae-miR399. TaPHO2-D1 was expressed much more abundantly than TaPHO2-A1 and -B1. The ion beam-induced deletion mutants were used to analyze the effects of TaPHO2s on phosphorus uptake and plant growth. The tapho2-a1, tapho2-b1 and tapho2-d1 mutants all had significant higher leaf Pi concentrations than did the wild type, with tapho2-d1 having the strongest effect, and tapho2-b1 the weakest. Two consecutive field experiments showed that knocking out TaPHO2-D1 reduced plant height and grain yield under both low and high phosphorus conditions. However, knocking out TaPHO2-A1 significantly increased phosphorus uptake and grain yield under low phosphorus conditions, with no adverse effect on grain yield under high phosphorus conditions. Our results indicated that TaPHO2s involved in phosphorus uptake and translocation, and molecular engineering TaPHO2 shows potential in improving wheat yield with less phosphorus fertilizer. PMID:27416927

  4. Investigation of olfactory function in a Panx1 knock out mouse model

    PubMed Central

    Kurtenbach, Stefan; Whyte-Fagundes, Paige; Gelis, Lian; Kurtenbach, Sarah; Brazil, Émerson; Zoidl, Christiane; Hatt, Hanns; Shestopalov, Valery I.; Zoidl, Georg

    2014-01-01

    Pannexin 1 (Panx1), the most extensively investigated member of a channel-forming protein family, is able to form pores conducting molecules up to 1.5 kDa, like ATP, upon activation. In the olfactory epithelium (OE), ATP modulates olfactory responsiveness and plays a role in proliferation and differentiation of olfactory sensory neurons (OSNs). This process continuously takes place in the OE, as neurons are replaced throughout the whole lifespan. The recent discovery of Panx1 expression in the OE raises the question whether Panx1 mediates ATP release responsible for modulating chemosensory function. In this study, we analyzed pannexin expression in the OE and a possible role of Panx1 in olfactory function using a Panx1−/− mouse line with a global ablation of Panx1. This mouse model has been previously used to investigate Panx1 functions in the retina and adult hippocampus. Here, qPCR, in-situ hybridization, and immunohistochemistry (IHC) demonstrated that Panx1 is expressed in axon bundles deriving from sensory neurons of the OE. The localization, distribution, and expression of major olfactory signal transduction proteins were not significantly altered in Panx1−/− mice. Further, functional analysis of Panx1−/− animals does not reveal any major impairment in odor perception, indicated by electroolfactogram (EOG) measurements and behavioral testing. However, ATP release evoked by potassium gluconate application was reduced in Panx1−/− mice. This result is consistent with previous reports on ATP release in isolated erythrocytes and spinal or lumbar cord preparations from Panx1−/− mice, suggesting that Panx1 is one of several alternative pathways to release ATP in the olfactory system. PMID:25309319

  5. Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways

    PubMed Central

    Choudhary, Mayur; Kazmin, Dmitri; Hu, Peng; Thomas, Russell S; McDonnell, Donald P; Malek, Goldis

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR−/− and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR−/− mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1+) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C–C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that

  6. Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways.

    PubMed

    Choudhary, Mayur; Kazmin, Dmitri; Hu, Peng; Thomas, Russell S; McDonnell, Donald P; Malek, Goldis

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR(-/-) and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR(-/-) mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1(+) ) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C-C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that

  7. The inflammation paradigm and coronary artery disease: What Celsus, Virchow and gene knock outs have taught us.

    PubMed

    Krishnaswamy, Guha

    2010-12-01

    Atherosclerotic vascular disease is a major cause of morbidity and mortality throughout the world. The clinical manifestations include coronary artery disease and myocardial infarction, cerebrovascular disease, renovascular disease and peripheral vascular disease. Initially considered a bland occlusive disease mediated to a great extent by lipids, atherosclerosis can now be considered an inflammatory disease, in its own right. This has led to a paradigm shift in disease management. We have come a long way since the time of Celsus, Galen, Virchow, Rokitansky and others when the components of the inflammatory cascade were first described. The development of mouse knock out models, improved molecular approaches to studying atheromatous blood vessels and development of sophisticated imaging and biomarker studies have enhanced our understanding of the molecular pathways in atherosclerosis. This brief review will attempt to weave together the historical, biochemical, immunological and molecular developments that have led to our current understanding of a deadly but treatable and potentially preventable disease.

  8. Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

    PubMed

    Kwon, Dae-Jin; Kim, Dong-Hwan; Hwang, In-Sul; Kim, Dong-Ern; Kim, Hyung-Joo; Kim, Jang-Seong; Lee, Kichoon; Im, Gi-Sun; Lee, Jeong-Woong; Hwang, Seongsoo

    2017-02-01

    Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT(-(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+)] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

  9. Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.

    PubMed

    Choi, Kimyung; Shim, Joohyun; Ko, Nayoung; Eom, Heejong; Kim, Jiho; Lee, Jeong-Woong; Jin, Dong-Il; Kim, Hyunil

    2017-04-01

    Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

  10. Loss of Bace2 in zebrafish affects melanocyte migration and is distinct from Bace1 knock out phenotypes.

    PubMed

    van Bebber, Frauke; Hruscha, Alexander; Willem, Michael; Schmid, Bettina; Haass, Christian

    2013-11-01

    Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid β-peptide (Aβ). Two proteases, β- and γ-secretase proteolytically generate Aβ from its precursor, the ß-amyloid precursor protein (APP). Inhibition of β-secretase, also referred to as beta-site APP cleaving enzyme (BACE1) or γ-secretase is therefore of prime interest for the development of amyloid-lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nuclease-mediated genome editing. bace1 mutants (bace1-/-) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1-/-. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1-/-. Double homozygous bace1-/-; bace2-/- fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 -/- associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo. Inhibition of BACE1 protease activity has therapeutic importance for Alzheimer's disease. Analysis of BACE1 and BACE2 knock-out zebrafish revealed that they exhibit distinct phenotypes. bace1 mutants display hypomyelination in the PNS and supernumerary neuromasts while in bace2 mutants the shape and migration of melanocytes is affected. These phenotypes are not further enhanced in the viable double mutants. Our data suggest that blocking BACE1 activity is a safe therapeutic approach.

  11. Knocking out ubiquitin proteasome system function in vivo and in vitro with genetically encodable tandem ubiquitin.

    PubMed

    Saeki, Y; Isono, E; Shimada, M; Kawahara, H; Yokosawa, H; Toh-E, A

    2005-01-01

    At present, the 26S proteasome-specific inhibitor is not available. We constructed polyubiquitin derivatives that contained a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. When these artificial polyubiquitins (tUbs, tandem ubiquitins) were overproduced in the wild-type yeast strain, growth was strongly inhibited, probably because of inhibition of the 26S proteasome. We also found that several substrates of the ubiquitin-proteasome pathway were stabilized by expressing tUbs in vivo. tUbs containing four units or more of the ubiquitin monomer were found to form a complex with the 26S proteasome. We showed that tUb bound to the 26S proteasome inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 (six-mer) messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

  12. Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

    SciTech Connect

    Li, Hong-Tao; Liu, Hua; Gao, Xiao-Shu; Zhang, Hongxia

    2009-05-08

    AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na{sup +} content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na{sup +} homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.

  13. [Study on material base of Carthamus tinctorius with antioxidant effect based on selective knock-out].

    PubMed

    Wang, Lin-Yan; Tang, Yu-Ping; Liu, Xin; Ge, Ya-Hui; Li, Shu-Jiao; Shang, Er-Xin; Duan, Jin-Ao

    2014-04-01

    To establish a method for studying efficacious materials of traditional Chinese medicines from an overall perspective. Carthamus tinctorius was taken the example. Its major components were depleted by preparing liquid chromatography. Afterwards, the samples with major components depleted were evaluated for their antioxidant effect, so as to compare and analyze the major efficacious materials of C. tinctorius with antioxidant activity and the contributions. Seven major components were depleted from C. tinctorius samples, and six of them were identified with MS data and control comparison. After all of the samples including depleted materials are compared and evaluated for their antioxidant effect, the findings showed that hydroxysafflor yellow A, anhydrosafflor yellow B and 6-hydroxykaempferol-3, 6-di-O-glucoside-7-O-glucuronide were the major efficacious materials. This study explored a novel and effective method for studying efficacious materials of traditional Chinese medicines. Through this method, we could explain the direct and indirect contributions of different components to the efficacy of traditional Chinese medicines, and make the efficacious material expression of traditional Chinese medicines clearer.

  14. 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.

  15. Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus).

    PubMed

    Choi, Seung Hyuk; Kim, Ki Hong

    2011-07-01

    Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10⁶ fold increase of LD₅₀ dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10⁷ CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10⁶ or 10⁵ CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10⁶-10⁷ CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10⁶-10

  16. Knocking out the dopamine reuptake transporter (DAT) does not change the baseline brain arachidonic acid signal in the mouse

    PubMed Central

    Ramadan, Epolia; Chang, Lisa; Chen, Mei; Ma, Kaizong; Hall, F. Scott; Uhl, George R.; Rapoport, Stanley I.; Basselin, Mireille

    2012-01-01

    Background Dopamine transporter (DAT) homozygous knockout (DAT−/−) mice have a 10-fold higher extracellular DA concentration in the caudate-putamen and nucleus accumbens than do wildtype (DAT+/+) mice, but show reduced presynaptic DA synthesis and fewer postsynaptic D2 receptors. One aspect of neurotransmission involves DA binding to postsynaptic D2-like receptors coupled to cytosolic phospholipase A2 (cPLA2), releasing second messenger arachidonic acid (AA) from synaptic membrane phospholipid. We hypothesized that tonic overactivation of D2-like receptors in DAT−/− mice due to elevated DA would not increase brain AA signaling, because of compensatory downregulation of postsynaptic signaling mechanisms. Methods [1-14C]AA was infused intravenously for 3 min in unanesthetized DAT+/+, heterozygous (DAT+/−) and DAT−/− mice. AA incorporation coefficients k* and rates Jin, markers of AA metabolism and signaling, were imaged in 83 brain regions using quantitative autoradiography brain cPLA2-IV activity also was measured. Results Neither k* nor Jin for AA in any brain region, or in brain cPLA2-IV activity, differed significantly between DAT−/−, DAT+/− and DAT+/+ mice. Conclusions These results differ from reported increases in k* and Jin for AA, and brain cPLA2 expression, in serotonin reuptake transporter (5-HTT) knockout mice, and suggest that postsynaptic dopaminergic neurotransmission mechanisms involving AA are downregulated despite elevated DA in DAT−/− mice. PMID:22376027

  17. COMPARATIVE HEPATIC EFFECTS OF PERFLUOROOCTANOIC ACID AND WY 14,643 IN PPARÁ KNOCKED OUT AND WILD-TYPE MICE

    EPA Science Inventory

    Perfluorooctanoic acid (PFOA) is a fluorinated organic chemical widely used in consumer and industrial products. Its persistence in the environment and presence in humans and wildlife have raised considerable concerns. PFOA induces liver tumors in rodents, which is thought to be ...

  18. COMPARATIVE HEPATIC EFFECTS OF PERFLUOROOCTANOIC ACID AND WY 14,643 IN PPARÁ KNOCKED OUT AND WILD-TYPE MICE

    EPA Science Inventory

    Perfluorooctanoic acid (PFOA) is a fluorinated organic chemical widely used in consumer and industrial products. Its persistence in the environment and presence in humans and wildlife have raised considerable concerns. PFOA induces liver tumors in rodents, which is thought to be ...

  19. 1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out

    PubMed Central

    2014-01-01

    Background 1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579. Results Genetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged. Conclusion Due to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production. PMID:24885849

  20. Reconstructing gene regulatory networks from knock-out data using Gaussian Noise Model and Pearson Correlation Coefficient.

    PubMed

    Mohamed Salleh, Faridah Hani; Arif, Shereena Mohd; Zainudin, Suhaila; Firdaus-Raih, Mohd

    2015-12-01

    A gene regulatory network (GRN) is a large and complex network consisting of interacting elements that, over time, affect each other's state. The dynamics of complex gene regulatory processes are difficult to understand using intuitive approaches alone. To overcome this problem, we propose an algorithm for inferring the regulatory interactions from knock-out data using a Gaussian model combines with Pearson Correlation Coefficient (PCC). There are several problems relating to GRN construction that have been outlined in this paper. We demonstrated the ability of our proposed method to (1) predict the presence of regulatory interactions between genes, (2) their directionality and (3) their states (activation or suppression). The algorithm was applied to network sizes of 10 and 50 genes from DREAM3 datasets and network sizes of 10 from DREAM4 datasets. The predicted networks were evaluated based on AUROC and AUPR. We discovered that high false positive values were generated by our GRN prediction methods because the indirect regulations have been wrongly predicted as true relationships. We achieved satisfactory results as the majority of sub-networks achieved AUROC values above 0.5. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia

    PubMed Central

    Zhang, Qifeng; Smethurst, Elizabeth; Segonds-Pichon, Anne; Schrewe, Heinrich; Wakelam, Michael J. O.

    2016-01-01

    Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction. PMID:27658289

  2. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

    PubMed

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-07-15

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.

  3. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

    PubMed Central

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  4. The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

    PubMed Central

    Xiong, Qiuhong; Ünal, Can; Matthias, Jan; Steinert, Michael; Eichinger, Ludwig

    2015-01-01

    Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16− and ATG9−/16− cells and compare them to the previously reported ATG9− mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9− and ATG16− cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9−/16− double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9−/16− cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9−/16− cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes. PMID:25878144

  5. Comparative N-linked glycan analysis of wild-type and α1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches.

    PubMed

    Park, Hae-Min; Kim, Yoon-Woo; Kim, Kyoung-Jin; Kim, Young June; Yang, Yung-Hun; Jin, Jang Mi; Kim, Young Hwan; Kim, Byung-Gee; Shim, Hosup; Kim, Yun-Gon

    2015-01-31

    Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

  6. Analysis of the synergistic effect of glycyrrhizin and other constituents in licorice extract on lipopolysaccharide-induced nitric oxide production using knock-out extract.

    PubMed

    Uto, Takuhiro; Morinaga, Osamu; Tanaka, Hiroyuki; Shoyama, Yukihiro

    2012-01-06

    The pharmacological evidence for synergism between natural compounds is not fully elucidated. In this study, we investigated the synergistic function of one target compound in medicinal plant extract by using knock-out (KO) extract, which is one target compound-eliminated extract from whole crude extract. Licorice is the most important ingredient used in the traditional Chinese medicine (TCM) and the Japanese Kampo medicine, and one of the major active components of licorice is glycyrrhizin (GC). To identify the potential role of GC, we prepared GC-removed extract (GC-KO extract) from licorice extract (LE) using immunoaffinity column conjugated with anti-GC monoclonal antibody (MAb), which could eliminate 99.5% of GC from LE. LE inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264 murine macrophage cells. However, treatment of GC alone could not show the suppression of NO production and iNOS expression. Interestingly, the inhibitory effect of GC-KO extract was significantly attenuated compared with LE. Furthermore, the combined treatment with GC-KO extract and GC could improve the attenuated inhibition. Taken together, our results indicate that GC may exert synergistic suppression of iNOS expression when coexisting with the other constituents contained in LE, and KO extract is a useful approach for determination of real pharmacological functions of natural compound in the phytochemical mixture.

  7. The GAD65 knock out mouse - a model for GABAergic processes in fear- and stress-induced psychopathology.

    PubMed

    Müller, Iris; Çalışkan, Gürsel; Stork, Oliver

    2015-01-01

    The γ-amino butyric acid (GABA) synthetic enzyme glutamic acid decarboxylase (GAD)65 is critically involved in the activity-dependent regulation of GABAergic inhibition in the central nervous system. It is also required for the maturation of the GABAergic system during adolescence, a phase that is critical for the development of several neuropsychiatric diseases. Mice bearing a null mutation of the GAD65 gene develop hyperexcitability of the amygdala and hippocampus, and a phenotype of increased anxiety and pathological fear memory reminiscent of posttraumatic stress disorder. Although genetic association of GAD65 in human has not yet been reported, these findings are in line with observations of reduced GABAergic function in these brain regions of anxiety disorder patients. The particular value of GAD65(-/-) mice thus lies in modeling the effects of reduced GABAergic function in the mature nervous system. The expression of GAD65 and a second GAD isozyme, GAD67, are differentially regulated in response to stress in limbic brain areas suggesting that by controlling GABAergic inhibition these enzymes determine the vulnerability for the development of pathological anxiety and other stress-induced phenotypes. In fact, we could recently show that GAD65 haplodeficiency, which results in delayed postnatal increase of GABA levels, provides resilience to juvenile-stress-induced anxiety to GAD65(+/-) mice thus foiling the increased fear and anxiety in homozygous GAD65(-/-) mice.

  8. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

    PubMed

    McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

    2016-09-01

    The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Zinc finger nuclease knock-out of NADPH:cytochrome P450 oxidoreductase (POR) in human tumor cell lines demonstrates that hypoxia-activated prodrugs differ in POR dependence.

    PubMed

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B; Smaill, Jeff B; Patterson, Adam V; Guise, Christopher P; Wilson, William R

    2013-12-27

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

  10. Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence*

    PubMed Central

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B.; Smaill, Jeff B.; Patterson, Adam V.; Guise, Christopher P.; Wilson, William R.

    2013-01-01

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development. PMID:24196959

  11. Orexin/Hypocretin and Histamine: Distinct Roles in the Control of Wakefulness Demonstrated Using Knock-Out Mouse Models

    PubMed Central

    Anaclet, Christelle; Parmentier, Régis; Ouk, Koliane; Guidon, Gérard; Buda, Colette; Sastre, Jean-Pierre; Akaoka, Hidéo; Sergeeva, Olga A.; Yanagisawa, Masashi; Ohtsu, Hiroshi; Franco, Patricia; Haas, Helmut L.; Lin, Jian Sheng

    2009-01-01

    To determine the respective role played by orexin/hypocretin and histamine (HA) neurons in maintaining wakefulness (W), we characterized the behavioral and sleep-wake phenotypes of orexin(Ox) knockout(−/−) mice and compared them with those of histidine-decarboxylase(HDC, HA-synthesizing enzyme)−/−mice. While both mouse strains displayed sleep fragmentation and increased paradoxical sleep(PS), they presented a number of marked differences: 1) The PS-increase in HDC−/−mice was seen during lightness, whereas that in Ox−/−mice occurred during darkness; 2) Contrary to HDC−/−, Ox−/−mice had no W deficiency around lights-off, nor an abnormal EEG and responded to a new environment with increased W; 3) Only Ox−/−, but not HDC−/−mice, displayed narcolepsy and deficient W when faced with motor challenge. Thus, when placed on a wheel, WT, but not littermate Ox−/−mice, voluntarily spent their time in turning it and as a result, remained highly awake; this was accompanied by dense c-fos expression in many areas of their brains, including Ox-neurons in the dorsolateral hypothalamus. The W and motor deficiency of Ox−/−mice was due to the absence of Ox because intraventricular dosing of Ox-A restored their W amount and motor performance whereas SB-334867 (Ox1-receptor antagonist, i.p.) impaired W and locomotion of WT mice during the test. These data indicate that Ox, but not HA, promotes W through enhanced locomotion and suggest that HA and Ox neurons exert a distinct, but complementary and synergistic control of W: the neuropeptide being more involved in its behavioral aspects, whereas the amine is mainly responsible for its qualitative cognitive aspects and cortical-EEG activation. PMID:19923277

  12. Otoconia-Deficient Mice Show Selective Spatial Deficits

    PubMed Central

    Yoder, Ryan M.; Kirby, Seth L.

    2014-01-01

    Damage or inactivation of the vestibular system impairs performance on various spatial memory tasks, but few studies have attempted to disambiguate the roles of the semicircular canals and otolith organs in this performance. The present study tested the otolithic contribution to spatial working and reference memory by evaluating the performance of otoconia-deficient tilted mice on a radial arm maze and a Barnes maze. One radial arm maze task provided both intramaze and extramaze cues, whereas the other task provided only extramaze cues. The Barnes maze task provided only extramaze cues. On the radial arm maze, tilted mice performed similar to control mice when intramaze cues were available, but made more working and reference memory errors than control mice when only extramaze cues were available. On the Barnes maze task, control and tilted mice showed similar latency, distance, and errors during acquisition training. On the subsequent probe trial, both groups spent the greatest percentage of time in the goal quadrant, indicating they were able to use extramaze cues to guide their search. Overall, these results suggest signals originating in the otolith organs contribute to spatial memory, but are not necessary for all aspects of spatial performance. PMID:24802640

  13. Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

    PubMed Central

    2013-01-01

    Background Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. Results A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. Conclusions The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal

  14. A network-based approach for predicting Hsp27 knock-out targets in mouse skeletal muscles

    PubMed Central

    Kammoun, Malek; Picard, Brigitte; Henry-Berger, Joëlle; Cassar-Malek, Isabelle

    2013-01-01

    Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Here, we have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions, e.g. members of Hsp families (Hsp20, Cryab, Hsp70a1a, and Hsp90aa1), regulators of apoptosis (Fas, Chuk, and caspase-3), translation factors (Eif4E, and Eif4G1), cytoskeletal proteins (Desmin) and antioxidants (Sod1). The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness. PMID:24688716

  15. Designing and Cloning Molecular Constructs to Knock Out N-Acetylglucosamine Phosphatidylinositol De-N-Acetylase (GPI12) Gene in Leishmania major (MRHO/IR/75/ER)

    PubMed Central

    GHASEMI NEJAD ALMANI, Pooya; SHARIFI, Iraj; KAZEMI, Bahram; BABAEI, Zahra; BANDEHPOUR, Mojgan; SALARI, Samira; SAEDI DEZAKI, Ebrahim

    2016-01-01

    Background: Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major. Methods: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages. Results: The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots. Conclusion: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host. PMID:28127356

  16. Towards in vivo mutation analysis: knock-out of specific chlorophylls bound to the light-harvesting complexes of Arabidopsis thaliana - the case of CP24 (Lhcb6).

    PubMed

    Passarini, Francesca; Xu, Pengqi; Caffarri, Stefano; Hille, Jacques; Croce, Roberta

    2014-09-01

    In the last ten years, a large series of studies have targeted antenna complexes of plants (Lhc) with the aim of understanding the mechanisms of light harvesting and photoprotection. Combining spectroscopy, modeling and mutation analyses, the role of individual pigments in these processes has been highlighted in vitro. In plants, however, these proteins are associated with multiple complexes of the photosystems and function within this framework. In this work, we have envisaged a way to bridge the gap between in vitro and in vivo studies by knocking out in vivo pigments that have been proposed to play an important role in excitation energy transfer between the complexes or in photoprotection. We have complemented a CP24 knock-out mutant of Arabidopsis thaliana with the CP24 (Lhcb6) gene carrying a His-tag and with a mutated version lacking the ligand for chlorophyll 612, a specific pigment that in vitro experiments have indicated as the lowest energy site of the complex. Both complexes efficiently integrated into the thylakoid membrane and assembled into the PSII supercomplexes, indicating that the His-tag does not impair the organization in vivo. The presence of the His-tag allowed the purification of CP24-WT and of CP24-612 mutant in their native states. It is shown that CP24-WT coordinates 10 chlorophylls and 2 carotenoid molecules and has properties identical to those of the reconstituted complex, demonstrating that the complex self-assembled in vitro assumes the same folding as in the plant. The absence of the ligand for chlorophyll 612 leads to the loss of one Chl a and of lutein, again as in vitro, indicating the feasibility of the method. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

  17. Croton grewioides Baill. (Euphorbiaceae) Shows Antidiarrheal Activity in Mice

    PubMed Central

    da Silva, Anne Dayse Soares; de Melo e Silva, Karoline; Neto, José Clementino; Costa, Vicente Carlos de Oliveira; Pessôa, Hilzeth de Luna F.; Tavares, Josean Fechine; da Silva, Marcelo Sobral; Cavalcante, Fabiana de Andrade

    2016-01-01

    Based on chemotaxonomy, we decided to investigate the possible antidiarrheal activity in mice of a crude ethanolic extract obtained from aerial parts of Croton grewioides (CG-EtOH). We tested for any possible toxicity in rat erythrocytes and acute toxicity in mice. Antidiarrheal activity was assessed by determining the effect of CG-EtOH on defecation frequency, liquid stool, intestinal motility and intestinal fluid accumulation. CG-EtOH showed no in vitro cytotoxicity and was not orally lethal. In contrast, the extract given intraperitoneally (at 2000 mg/kg) was lethal, but only in females. CG-EtOH produced a significant and equipotent antidiarrheal activity, both in defecation frequency (ED50 = 106.0 ± 8.1 mg/kg) and liquid stools (ED50 = 105.0 ± 9.2 mg/kg). However, CG-EtOH (125 mg/kg) decreased intestinal motility by only 22.7% ± 4.4%. Moreover, extract markedly inhibited the castor oil-induced intestinal contents (ED50 = 34.6 ± 5.4 mg/kg). We thus conclude that CG-EtOH is not orally lethal and contains active principles with antidiarrheal activity, and this effect seems to involve mostly changes in intestinal secretion. SUMMARY CG-EtOH showed no in vitro cytotoxicity and was not orally lethal. In contrast, the extract given intraperitoneally (at 2000 mg/kg) was lethal, but only in females.CG-EtOH probably contains active metabolites with antidiarrheal activity.CG-EtOH reduced the frequency and number of liquid stools.Metabolites presents in the CG-EtOH act mainly by reducing intestinal fluid and, to a lesser extent, reducing intestinal motility. Abbreviations Used: CG-EtOH: crude ethanolic extract obtained from the aerial parts of C. grewioides; WHO: World Health Organization; ED50: dose of a drug that produces 50% of its maximum effect; Emax: maximum effect PMID:27365990

  18. Mice Lacking Brinp2 or Brinp3, or Both, Exhibit Behaviors Consistent with Neurodevelopmental Disorders

    PubMed Central

    Berkowicz, Susan R.; Featherby, Travis J.; Whisstock, James C.; Bird, Phillip I.

    2016-01-01

    Background: Brinps 1–3, and Astrotactins (Astn) 1 and 2, are members of the Membrane Attack Complex/Perforin (MACPF) superfamily that are predominantly expressed in the mammalian brain during development. Genetic variation at the human BRINP2/ASTN1 and BRINP1/ASTN2 loci has been implicated in neurodevelopmental disorders. We, and others, have previously shown that Brinp1−/− mice exhibit behavior reminiscent of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Method: We created Brinp2−/− mice and Brinp3−/− mice via the Cre-mediated LoxP system to investigate the effect of gene deletion on anatomy and behavior. Additionally, Brinp2−/−Brinp3−/− double knock-out mice were generated by interbreeding Brinp2−/− and Brinp3−/− mice. Genomic validation was carried out for each knock-out line, followed by histological, weight and behavioral examination. Brinp1−/−Brinp2−/−Brinp3−/− triple knock-out mice were also generated by crossing Brinp2/3 double knock-out mice with previously generated Brinp1−/− mice, and examined by weight and histological analysis. Results: Brinp2−/− and Brinp3−/− mice differ in their behavior: Brinp2−/− mice are hyperactive, whereas Brinp3−/− mice exhibit marked changes in anxiety-response on the elevated plus maze. Brinp3−/− mice also show evidence of altered sociability. Both Brinp2−/− and Brinp3−/− mice have normal short-term memory, olfactory responses, pre-pulse inhibition, and motor learning. The double knock-out mice show behaviors of Brinp2−/− and Brinp3−/− mice, without evidence of new or exacerbated phenotypes. Conclusion: Brinp3 is important in moderation of anxiety, with potential relevance to anxiety disorders. Brinp2 dysfunction resulting in hyperactivity may be relevant to the association of ADHD with chromosome locus 1q25.2. Brinp2−/− and Brinp3−/− genes do not compensate in the mammalian brain and likely have

  19. Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects.

    PubMed

    Henriques, Rossana; Jásik, Ján; Klein, Markus; Martinoia, Enrico; Feller, Urs; Schell, Jeff; Pais, Maria S; Koncz, Csaba

    2002-11-01

    IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

  20. [Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis].

    PubMed

    Liu, Lian; Zhang, Ran-Ran; Yang, Qian; Wang, Xu; Gui, Yong-Hao

    2017-06-25

    Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a(-/-)(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2a(-/-) zebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a(-/-)(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2a(-/-) zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.

  1. Broken or knocked out tooth

    MedlinePlus

    Cohenca N. Management of traumatic dental injuries. In: Torabinejad M, Walton, RE, Fouad AF, eds. Endodontics: Principles and Practice . 5th ed. St. Louis, MO: Elsevier Saunders; 2015:chap 11. Tinanoff N. Dental trauma. In: Kliegman ...

  2. [Biological characteristics of an Hog1 MAPK homologous gene FoHog1 knock-out mutant of Fusarium oxysporum f. sp. cubense].

    PubMed

    Mao, Chao; Chen, Pingya; Dai, Qingdong; Yang, Laying; Huang, Junsheng

    2014-11-04

    This study was aimed to obtain a mitogen-activated protein kinase (MAPK) gene namely FoHog1 from Fusarium oxysporum f. sp. cubense and to verify its function. We amplified FoHog1 gene by PCR and RT-PCR methods and analyzed it through bioinformatics method. PEG-mediated protoplast transformation was used to create the deletion mutants of FoHog1 gene. We analyzed different biological characteristics between knock-out strain and wild-type strain. FoHog1 gene encoding a putative protein of 357 amino acids and its genetic relationship with different Fusarium' s protein. Compared with the wild-type strain, FoHog1 deletion mutants have loose hyphae colony, less spores production, lower dry weight of hyphae and more sensitive to temperature, pH and osmotic stress. FoHog1 deletion mutants also have reduced colonization ability compared with the wild-type strain. FoHog1 gene participated in mycelial growth, sporulation, catabolism of sodium acetate and ammonium chloride, osmotic stress response and pathogenic process with Fusarium oxysporum f. sp. cubense Race 4.

  3. [Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system].

    PubMed

    Yang, Yue; Wang, Haicheng; Xu, Shuyu; Peng, Jing; Jiang, Jiuhui; Li, Cuiying

    2015-08-01

    To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells, via the clustered regularly interspaced short palindromic repeats (CRISPR)/ associated proteins (Cas) system. One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream. Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells. PCR and DNA sequence were used to testify the knockout cells, and the monoclones of EDA absent SACC cells were selected (A+C-2, A+C-6, B+C-10). CCK-8 cell proliferation and invasion was then tested in control group and the experimental group. The sgRNA was successfully linked into PX-330 plasmid. Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out, and the knockdown efficiency was above 70%, but the total amount of fibronectin did not change significantly. Three monoclones of EDA absent SACC- 83 cells were successfully selected with diminished migration and proliferation. The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.

  4. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters

    PubMed Central

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G.; Gannavaram, Sreenivas; Nakhasi, Hira L.

    2016-01-01

    Background Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. Methodology/Principal Findings To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Conclusions Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or

  5. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters.

    PubMed

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G; Gannavaram, Sreenivas; Nakhasi, Hira L

    2016-01-01

    Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

  6. Production of superoxide from photosystem II-light harvesting complex II supercomplex in STN8 kinase knock-out rice mutants under photoinhibitory illumination.

    PubMed

    Poudyal, Roshan Sharma; Nath, Krishna; Zulfugarov, Ismayil S; Lee, Choon-Hwan

    2016-09-01

    When phosphorylation of Photosystem (PS) II core proteins is blocked in STN8 knock-out mutants of rice (Oryza sativa) under photoinhibitory illumination, the mobilization of PSII supercomplex is prevented. We have previously proposed that more superoxide (O2(-)) is produced from PSII in the mutant (Nath et al., 2013, Plant J. 76, 675-686). Here, we clarify the type and site for the generation of reactive oxygen species (ROS). Using both histochemical and fluorescence probes, we observed that, compared with wild-type (WT) leaves, levels of ROS, including O2(-) and hydrogen peroxide (H2O2), were increased when leaves from mutant plants were illuminated with excess light. However, singlet oxygen production was not enhanced under such conditions. When superoxide dismutase was inhibited, O2(-) production was increased, indicating that it is the initial event prior to H2O2 production. In thylakoids isolated from WT leaves, kinase was active in the presence of ATP, and spectrophotometric analysis of nitrobluetetrazolium absorbance for O2(-) confirmed that PSII-driven superoxide production was greater in the mutant thylakoids than in the WT. This contrast in levels of PSII-driven superoxide production between the mutants and the WT plants was confirmed by conducting protein oxidation assays of PSII particles from osstn8 leaves under strong illumination. Those assays also demonstrated that PSII-LHCII supercomplex proteins were oxidized more in the mutant, thereby implying that PSII particles incur greater damage even though D1 degradation during PSII-supercomplex mobilization is partially blocked in the mutant. These results suggest that O2(-) is the major form of ROS produced in the mutant, and that the damaged PSII in the supercomplex is the primary source of O2(-).

  7. Mouse Pet-1 knock-out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness

    PubMed Central

    Schaefer, Tori L.; Vorhees, Charles V.; Williams, Michael T.

    2009-01-01

    Serotonin (5-HT) is involved in many developmental processes and influences behaviors including anxiety, aggression, and cognition. Disruption of the serotonergic system has been implicated in human disorders including autism, depression, schizophrenia, and ADHD. Although pharmacological, neurotoxin, and dietary manipulation of 5-HT and tryptophan hydroxylase has added to our understanding of the serotonergic system, the results are complicated by multiple factors. A newly identified ETS domain transcription factor, Pet-1, has direct control of major aspects of 5-HT neuronal development. Pet-1 is the only known factor that is restricted in the brain to 5-HT neurons during development and adulthood and exerts dominant control over 5-HT neuronal phenotype. Disruption of Pet-1 produces an ∼80% loss of 5-HT neurons and content and results in increased aggression in male Pet-1-/- mice (Hendricks et al., 2003). We hypothesized that Pet-1-/- mice would also exhibit changes in anxiety and cognition. Pet-1-/- mice were hypoactive which may have affected the observed lack of anxious behavior in the elevated zero maze and light-dark test. Pet-1-/- mice, however, were more defensive during marble burying and showed acoustic startle hyper-reactivity. No deficits in spatial, egocentric, or novel object recognition learning were found in Pet-1-/- mice. These findings were unexpected given that 5-HT depleting drugs given to adult or developing animals result in learning deficits (Mazer et al., 1997;Morford et al., 2002;Vorhees et al., 2007). Lack of differences may be the result of compensatory mechanisms in reaction to a constitutive knockout of Pet-1 or 5-HT may not be as important in learning and memory as previously suspected. PMID:19786075

  8. The alpha-fetoprotein knock-out mouse model suggests that parental behavior is sexually differentiated under the influence of prenatal estradiol.

    PubMed

    Keller, Matthieu; Pawluski, Jodi L; Brock, Olivier; Douhard, Quentin; Bakker, Julie

    2010-04-01

    In rodent species, sexual differentiation of the brain for many reproductive processes depends largely on estradiol. This was recently confirmed again by using the alpha-fetoprotein knockout (AFP-KO) mouse model, which lacks the protective actions of alpha-fetoprotein against maternal estradiol and as a result represents a good model to determine the contribution of prenatal estradiol to the sexual differentiation of the brain and behavior. Female AFP-KO mice were defeminized and masculinized with regard to their neuroendocrine responses as well as sexual behavior. Since parental behavior is also strongly sexually differentiated in mice, we used the AFP-KO mouse model here to ask whether parental responses are differentiated prenatally under the influence of estradiol. It was found that AFP-KO females showed longer latencies to retrieve pups to the nest and also exhibited lower levels of crouching over the pups in the nest in comparison to WT females. In fact, they resembled males (WT and AFP-KO). Other measures of maternal behavior, for example the incidence of infanticide, tended to be higher in AFP-KO females than in WT females but this increase failed to reach statistical significance. The deficits observed in parental behavior of AFP-KO females could not be explained by any changes in olfactory function, novelty recognition or anxiety. Thus our results suggest that prenatal estradiol defeminizes the parental brain in mice.

  9. Narp knockout mice show normal reactivity to novelty but attenuated recovery from neophobia.

    PubMed

    Blouin, Ashley M; Lee, Jongah J; Tao, Bo; Smith, Dani R; Johnson, Alexander W; Baraban, Jay M; Reti, Irving M

    2013-11-15

    Narp knockout (KO) mice demonstrate cognitive inflexibility and addictive behavior, which are associated with abnormal reactivity to a novel stimulus. To assess reactivity to novelty, we tested Narp KO and wild-type (WT) mice on a neophobia procedure. Both Narp KO and WT mice showed a similar decrease in consumption upon initial exposure to a novel flavor, but Narp KO mice did not increase consumption with subsequent exposures to the novel flavor like the WT mice. Therefore, Narp KO mice do not have abnormal reactivity to novelty but show deficits in adapting behavior to reflect the updated value of a stimulus.

  10. Reduced acute nociception and chronic pain in Shank2-/- mice.

    PubMed

    Ko, Hyoung-Gon; Oh, Seog-Bae; Zhuo, Min; Kaang, Bong-Kiun

    2016-01-01

    Autism spectrum disorder is a debilitating mental illness and social issue. Autism spectrum disorder patients suffer from social isolation, cognitive deficits, compulsive behavior, and sensory deficits, including hyposensitivity to pain. However, recent studies argued that autism spectrum disorder patients show physiological pain response and, in some cases, even extremely intense pain response to harmless stimulation. Recently, Shank gene family was reported as one of the genetic risk factors of autism spectrum disorder. Thus, in this study, we used Shank2(-) (/) (-) (Shank2 knock-out, KO) mice to investigate the controversial pain sensitivity issue and found that Shank2 KO mice showed reduced tactile perception and analgesia to chronic pain.

  11. Conditional knock-out reveals a requirement for O-linked N-Acetylglucosaminase (O-GlcNAcase) in metabolic homeostasis.

    PubMed

    Keembiyehetty, Chithra; Love, Dona C; Harwood, Katryn R; Gavrilova, Oksana; Comly, Marcella E; Hanover, John A

    2015-03-13

    O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear. Here, we report a conditional disruption of the Oga gene in the mouse. The resulting homozygous Oga null (KO) animals lack OGA enzymatic activity and exhibit elevated levels of the O-GlcNAc modification. The Oga KO animals showed nearly complete perinatal lethality associated with low circulating glucose and low liver glycogen stores. Defective insulin-responsive GSK3β phosphorylation was observed in both heterozygous (HET) and KO Oga animals. Although Oga HET animals were viable, they exhibited alterations in both transcription and metabolism. Transcriptome analysis using mouse embryonic fibroblasts revealed deregulation in the transcripts of both HET and KO animals specifically in genes associated with metabolism and growth. Additionally, metabolic profiling showed increased fat accumulation in HET and KO animals compared with WT, which was increased by a high fat diet. Reduced insulin sensitivity, glucose tolerance, and hyperleptinemia were also observed in HET and KO female mice. Notably, the respiratory exchange ratio of the HET animals was higher than that observed in WT animals, indicating the preferential utilization of glucose as an energy source. These results suggest that the loss of mouse OGA leads to defects in metabolic homeostasis culminating in obesity and insulin resistance.

  12. Conditional Knock-out Reveals a Requirement for O-Linked N-Acetylglucosaminase (O-GlcNAcase) in Metabolic Homeostasis*

    PubMed Central

    Keembiyehetty, Chithra; Love, Dona C.; Harwood, Katryn R.; Gavrilova, Oksana; Comly, Marcella E.; Hanover, John A.

    2015-01-01

    O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear. Here, we report a conditional disruption of the Oga gene in the mouse. The resulting homozygous Oga null (KO) animals lack OGA enzymatic activity and exhibit elevated levels of the O-GlcNAc modification. The Oga KO animals showed nearly complete perinatal lethality associated with low circulating glucose and low liver glycogen stores. Defective insulin-responsive GSK3β phosphorylation was observed in both heterozygous (HET) and KO Oga animals. Although Oga HET animals were viable, they exhibited alterations in both transcription and metabolism. Transcriptome analysis using mouse embryonic fibroblasts revealed deregulation in the transcripts of both HET and KO animals specifically in genes associated with metabolism and growth. Additionally, metabolic profiling showed increased fat accumulation in HET and KO animals compared with WT, which was increased by a high fat diet. Reduced insulin sensitivity, glucose tolerance, and hyperleptinemia were also observed in HET and KO female mice. Notably, the respiratory exchange ratio of the HET animals was higher than that observed in WT animals, indicating the preferential utilization of glucose as an energy source. These results suggest that the loss of mouse OGA leads to defects in metabolic homeostasis culminating in obesity and insulin resistance. PMID:25596529

  13. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

    PubMed

    Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-11-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. © 2015 Wiley Periodicals, Inc.

  14. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis

    PubMed Central

    Wu, Lian; Wang, Feng; Donly, Kevin J.; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2016-01-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  15. Comparative Analysis of the Effects of Hydroxysafflor Yellow A and Anhydrosafflor Yellow B in Safflower Series of Herb Pairs Using Prep-HPLC and a Selective Knock-Out Approach.

    PubMed

    Qu, Cheng; Wang, Lin-Yan; Jin, Wen-Tao; Tang, Yu-Ping; Jin, Yi; Shi, Xu-Qin; Shang, Li-Li; Shang, Er-Xin; Duan, Jin-Ao

    2016-11-06

    The flower of Carthamus tinctorius L. (Carthami Flos, safflower), important in traditional Chinese medicine (TCM), is known for treating blood stasis, coronary heart disease, hypertension, and cerebrovascular disease in clinical and experimental studies. It is widely accepted that hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) are the major bioactive components of many formulae comprised of safflower. In this study, selective knock-out of target components such as HSYA and ASYB by using preparative high performance liquid chromatography (prep-HPLC) followed by antiplatelet and anticoagulation activities evaluation was used to investigate the roles of bioactive ingredients in safflower series of herb pairs. The results showed that both HSYA and ASYB not only played a direct role in activating blood circulation, but also indirectly made a contribution to the total bioactivity of safflower series of herb pairs. The degree of contribution of HSYA in the safflower and its series herb pairs was as follows: Carthami Flos-Ginseng Radix et Rhizoma Rubra (CF-GR) > Carthami Flos-Sappan Lignum (CF-SL) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Astragali Radix (CF-AR) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Glycyrrhizae Radix et Rhizoma (CF-GL) > Carthami Flos-Salviae Miltiorrhizae Radix et Rhizoma (CF-SM) > Carthami Flos (CF), and the contribution degree of ASYB in the safflower and its series herb pairs: CF-GL > CF-PS > CF-AS > CF-SL > CF-SM > CF-AR > CF-GR > CF. So, this study provided a significant and effective approach to elucidate the contribution of different herbal components to the bioactivity of the herb pair, and clarification of the variation of herb-pair compatibilities. In addition, this study provides guidance for investigating the relationship between herbal compounds and the bioactivities of herb pairs. It also provides a scientific basis for reasonable clinical applications and new drug

  16. Mice deficient in PAPP-A show resistance to the development of diabetic nephropathy.

    PubMed

    Mader, Jessica R; Resch, Zachary T; McLean, Gary R; Mikkelsen, Jakob H; Oxvig, Claus; Marler, Ronald J; Conover, Cheryl A

    2013-10-01

    We investigated pregnancy-associated plasma protein-A (PAPP-A) in diabetic nephropathy. Normal human kidney showed specific staining for PAPP-A in glomeruli, and this staining was markedly increased in diabetic kidney. To assess the possible contribution of PAPP-A in the development of diabetic nephropathy, we induced diabetes with streptozotocin in 14-month-old WT and Papp-A knockout (KO) mice. Renal histopathology was evaluated after 4 months of stable hyperglycemia. Kidneys from diabetic WT mice showed multiple abnormalities including thickening of Bowman's capsule (100% of mice), increased glomerular size (80% of mice), tubule dilation (80% of mice), and mononuclear cell infiltration (90% of mice). Kidneys of age-matched non-diabetic WT mice had similar evidence of tubule dilation and mononuclear cell infiltration to those of diabetic WT mice, indicating that these changes were predominantly age-related. However, thickened Bowman's capsule and increased glomerular size appeared specific for the experimental diabetes. Kidneys from diabetic Papp-A KO mice had significantly reduced or no evidence of changes in Bowman's capsule thickening and glomerular size. There was also a shift to larger mesangial area and increased macrophage staining in diabetic WT mice compared with Papp-A KO mice. In summary, elevated PAPP-A expression in glomeruli is associated with diabetic nephropathy in humans and absence of PAPP-A is associated with resistance to the development of indicators of diabetic nephropathy in mice. These data suggest PAPP-A as a potential therapeutic target for diabetic nephropathy.

  17. Developmental Toxicity of Perfluorononanoic Acid in the Wild-Type and PPAR-alpha Knock-out Mouse After Gestational Exposure

    EPA Science Inventory

    Perfluorononanoic acid (PFNA) is a perfluoroalkyl acid detected in the environment and in tissues of humans and wildlife, and its concentration in human serum has increased in the past few years. PFNA negatively affects development and survival of CD1 mice and activates peroxisom...

  18. Developmental Toxicity of Perfluorononanoic Acid in the Wild-Type and PPAR-alpha Knock-out Mouse After Gestational Exposure

    EPA Science Inventory

    Perfluorononanoic acid (PFNA) is a perfluoroalkyl acid detected in the environment and in tissues of humans and wildlife, and its concentration in human serum has increased in the past few years. PFNA negatively affects development and survival of CD1 mice and activates peroxisom...

  19. Analysis of lymphocytes in, and host environment of, mice showing conditioned immunosuppression to cyclophosphamide

    SciTech Connect

    Gorczynski, R.M.

    1987-03-01

    Mice were subjected to repeated exposures to cyclophosphamide: saccharin (conditioned) or cyclophosphamide:saccharin followed by saccharin only (conditioned:extinguished). Animals in the former group but not the latter subsequently showed diminished IgG antibody-forming cells (AFC) after challenge with sheep red blood cells followed by reexposure to immunologically inert cues (saccharin). When these animals were used as irradiated recipients of syngeneic spleen lymphocytes, reconstituted irradiated conditioned mice showed augmented IgG AFC on transfer of naive spleen cells and reexposure to saccharin. The expected diminished IgG AFC response was seen when cells from conditioned mice were transferred. However, the latter cells gave augmented IgG AFC when transferred to naive irradiated mice. Both of the effects seen with cells from conditioned animals (increased IgG AFC in control recipients; decreased IgG AFC in conditioned mice reexposed to saccharin) were regulated by adoptively transferred T cells in the spleen cell population.

  20. Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

    PubMed

    Hutchens, Steven; Liu, Chunyi; Jursa, Thomas; Shawlot, William; Chaffee, Beth K; Yin, Weiling; Gore, Andrea C; Aschner, Michael; Smith, Donald R; Mukhopadhyay, Somshuvra

    2017-06-09

    Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization.

    PubMed

    Rolán, Hortensia García; Tsolis, Renée M

    2007-06-01

    The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.

  2. BDNF-Deficient Mice Show Reduced Psychosis-Related Behaviors Following Chronic Methamphetamine.

    PubMed

    Manning, Elizabeth E; Halberstadt, Adam L; van den Buuse, Maarten

    2016-04-01

    One of the most devastating consequences of methamphetamine abuse is increased risk of psychosis. Brain-derived neurotrophic factor has been implicated in both psychosis and neuronal responses to methamphetamine. We therefore examined persistent psychosis-like behavioral effects of methamphetamine in brain-derived neurotrophic factor heterozygous mice. Mice were chronically treated with methamphetamine from 6 to 9 weeks of age, and locomotor hyperactivity to an acute D-amphetamine challenge was tested in photocell cages after a 2-week withdrawal period. Methamphetamine-treated wild-type mice, but not brain-derived neurotrophic factor heterozygous mice, showed locomotor sensitization to acute 3mg/kg D-amphetamine. Qualitative analysis of exploration revealed tolerance to D-amphetamine effects on entropy in methamphetamine-treated brain-derived neurotrophic factor heterozygous mice, but not wild-type mice. Chronic methamphetamine exposure induces contrasting profiles of behavioral changes in wild-type and brain-derived neurotrophic factor heterozygous mice, with attenuation of behaviors relevant to psychosis in methamphetamine-treated brain-derived neurotrophic factor heterozygous mice. This suggests that brain-derived neurotrophic factor signalling changes may contribute to development of psychosis in methamphetamine users. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  3. Mice lacking the galanin gene show decreased sensitivity to nicotine conditioned place preference.

    PubMed

    Neugebauer, Nichole M; Henehan, Robert M; Hales, Claire A; Picciotto, Marina R

    2011-03-01

    Previous work has indicated that the neuropeptide galanin decreases sensitivity to the rewarding effects of morphine and cocaine, but increases alcohol drinking. The aim of the current study was to examine the role of galanin signaling in nicotine reward by testing the effects of nicotine in mice lacking galanin peptide (GAL-/-) as compared to wild-type (GAL+/+) controls. Using an unbiased, three-chamber conditioned place preference (CPP) paradigm the dose-response function for nicotine CPP was tested in GAL-/- and GAL+/+ mice. Since activation of extracellular signal-related kinase (ERK2) is involved in the rewarding effects of several classes of drugs of abuse, we then measured the level of ERK2 phosphorylation in the nucleus accumbens shell (NACsh) and core (NACco) of GAL-/- and GAL+/+ mice following re-exposure to the CPP chamber previously paired with nicotine as a marker of mesolimbic system activation. Finally, we examined whether acute nicotine administration affects ERK2 activity in GAL-/- and GAL+/+ mice. GAL-/- mice required a higher dose of nicotine to induce a significant CPP compared to GAL+/+ mice. In the conditioning groups showing significant expression of nicotine CPP, only GAL+/+ mice showed ERK2 activation in the NACsh. This suggests that the nicotine CPP observed in GAL+/+ mice resulted in differential recruitment of ERK signaling in the NACsh compared to GAL-/- mice. In addition, no activation of ERK2 was observed following acute nicotine administration in either genotype. These data, along with prior results, suggest that galanin alters sensitivity to drugs of abuse differentially, with morphine, cocaine and amphetamine place preference suppressed, and nicotine and alcohol preference increased, by galanin signaling.

  4. Localized brain differences in Arc expression between mice showing low vs. high propensity to ethanol sensitization.

    PubMed

    Nona, Christina N; Lam, Marcus; Nobrega, José N

    2016-03-01

    Behavioral sensitization to ethanol (EtOH) manifests as a progressive and enduring increase in locomotor activity with repeated drug exposure. However, not all mice sensitize to EtOH and the neuronal mechanisms mediating vulnerability and resistance to EtOH sensitization remain unclear. We examined regional brain expression of the immediate early gene activity-regulated cytoskeleton-associated protein (Arc) in order to identify brain areas in which neuroplastic changes may contribute to the development and expression of EtOH sensitization. Male DBA/2J mice received 5 biweekly injections of EtOH (2.2g/kg, i.p.) or saline (SAL). They were categorized as high- (HS) or low-sensitized (LS) on the basis of final locomotor activity scores. In both LS and HS mice sacrificed after the last sensitization injection, Arc expression was decreased throughout the brain in comparison to SAL animals. A similar pattern was seen in mice sacrificed after an EtOH challenge two weeks after the last sensitization injection. However in this cohort, Arc expression was significantly increased in the central amygdala (CeA) in LS mice and in SAL mice receiving EtOH for the first time. No significant increases in Arc expression were seen in brains of sensitized (HS) animals. These results indicate an acute EtOH challenge results in different patterns of Arc expression in brains of LS, HS, and SAL mice. The dramatic increases in Arc expression in the CeA in LS and SAL mice showing little or no behavioral activation suggests that neural activity in this region may serve to inhibit the stimulant effects of EtOH. The observation that HS mice do not show increases in Arc expression with an EtOH challenge suggests the possibility that increased tolerance to the Arc-inducing effects of EtOH may be a factor in behavioral sensitization.

  5. Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI

    PubMed Central

    Gallagher, Joseph J.; Zhang, Xiaowei; Ziomek, Greg; Jacobs, Russell E.; Bearer, Elaine L.

    2012-01-01

    Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer’s disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n = 12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn2+ transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP −/− mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7 hours and reduced transport to the contralateral hippocampus after 25 hours; axonal transport deficits in the APP −/− animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning. PMID:22500926

  6. Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI.

    PubMed

    Gallagher, Joseph J; Zhang, Xiaowei; Ziomek, Gregory J; Jacobs, Russell E; Bearer, Elaine L

    2012-04-15

    Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer's disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n=12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn(2+) transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP-/- mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7h and reduced transport to the contralateral hippocampus after 25 h; axonal transport deficits in the APP-/- animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning.

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

    PubMed

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

    2017-01-01

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

  8. Mice lacking adenylyl cyclase type 5 (AC5) show increased ethanol consumption and reduced ethanol sensitivity.

    PubMed

    Kim, Kyoung-Shim; Kim, Hannah; Baek, In-Sun; Lee, Ko-Woon; Han, Pyung-Lim

    2011-05-01

    The adenylyl cyclase (AC)/cAMP system is believed to be a key component in regulating alcohol-drinking behavior. It was reported that adenylyl cyclase-5 (AC5) is expressed widely in the brain, with a preferential concentration in the dorsal striatum and nucleus accumbens, brain regions which are important for addiction and emotion. AC5 has been shown to be an essential mediator of morphine addiction and dopamine receptor function; however, it remains unknown whether or not AC5 plays a role in ethanol preference and sensitivity in animals. This work was carried out to determine the role of AC5 in alcohol consumption and the hypnotic response to alcohol using AC5 knockout (KO) mice. In the test for ethanol preference employing a two-bottle free-choice paradigm, AC5 KO mice showed increased ethanol consumption and preference compared with the wild-type mice. Ethanol-induced hypothermia was weakly reduced in AC5 KO mice. AC5 KO mice exhibited sedation/behavioral sleep to high-dose ethanol, but their responses were greatly suppressed compared with the wild-type mice. These results suggest that AC5 is an important signaling molecule regulating alcohol sensitivity and preference in animals. These data provide critical information for AC5 activation as a candidate target for the treatment of alcoholism.

  9. The cl2/dro1/ccdc80 null mice develop thyroid and ovarian neoplasias.

    PubMed

    Leone, Vincenza; Ferraro, Angelo; Schepis, Filippo; Federico, Antonella; Sepe, Romina; Arra, Claudio; Langella, Concetta; Palma, Giuseppe; De Lorenzo, Carlo; Troncone, Giancarlo; Masciullo, Valeria; Scambia, Giovanni; Fusco, Alfredo; Pallante, Pierlorenzo

    2015-02-28

    We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80(-/-) mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80(-/-) mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Heterozygous Che-1 KO mice show deficiencies in object recognition memory persistence.

    PubMed

    Zalcman, Gisela; Corbi, Nicoletta; Di Certo, Maria Grazia; Mattei, Elisabetta; Federman, Noel; Romano, Arturo

    2016-10-06

    Transcriptional regulation is a key process in the formation of long-term memories. Che-1 is a protein involved in the regulation of gene transcription that has recently been proved to bind the transcription factor NF-κB, which is known to be involved in many memory-related molecular events. This evidence prompted us to investigate the putative role of Che-1 in memory processes. For this study we newly generated a line of Che-1(+/-) heterozygous mice. Che-1 homozygous KO mouse is lethal during development, but Che-1(+/-) heterozygous mouse is normal in its general anatomical and physiological characteristics. We analyzed the behavioral characteristic and memory performance of Che-1(+/-) mice in two NF-κB dependent types of memory. We found that Che-1(+/-) mice show similar locomotor activity and thigmotactic behavior than wild type (WT) mice in an open field. In a similar way, no differences were found in anxiety-like behavior between Che-1(+/-) and WT mice in an elevated plus maze as well as in fear response in a contextual fear conditioning (CFC) and object exploration in a novel object recognition (NOR) task. No differences were found between WT and Che-1(+/-) mice performance in CFC training and when tested at 24h or 7days after training. Similar performance was found between groups in NOR task, both in training and 24h testing performance. However, we found that object recognition memory persistence at 7days was impaired in Che-1(+/-) heterozygous mice. This is the first evidence showing that Che-1 is involved in memory processes.

  11. Mice carrying a CAR-2 null allele lack carbonic anhydrase II immunohistochemically and show vascular calcification.

    PubMed Central

    Spicer, S. S.; Lewis, S. E.; Tashian, R. E.; Schulte, B. A.

    1989-01-01

    Mutant mice reported to lack carbonic anhydrase isoenzyme II (CA II) have been examined here for immunocytochemical evidence of CA II and for histopathologic change. All histologic sites that immunostain for CA II in a wide range of organs in normal mice failed to show such immunoreactivity in the homozygous mutants. The CA II-deficient mice differed from controls in evidencing an age dependent medial calcification of small arteries in a number of organs. The male genital tract revealed the most extensive arterial calcinosis and males were possibly more affected in general than females. One or another Car-2n/Car-2n mouse showed changes additionally in uterus, small bowel, lymph nodes, or renal pelvis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 PMID:2495727

  12. Pancreas-specific aquaporin 12 null mice showed increased susceptibility to caerulein-induced acute pancreatitis.

    PubMed

    Ohta, Eriko; Itoh, Tomohiro; Nemoto, Tomomi; Kumagai, Jiro; Ko, Shigeru B H; Ishibashi, Kenichi; Ohno, Mayuko; Uchida, Keiko; Ohta, Akihito; Sohara, Eisei; Uchida, Shinichi; Sasaki, Sei; Rai, Tatemitsu

    2009-12-01

    Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe pathological damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation.

  13. Differential proteome-metabolome profiling of YCA1-knock-out and wild type cells reveals novel metabolic pathways and cellular processes dependent on the yeast metacaspase.

    PubMed

    Ždralević, Maša; Longo, Valentina; Guaragnella, Nicoletta; Giannattasio, Sergio; Timperio, Anna Maria; Zolla, Lello

    2015-06-01

    The yeast Saccharomyces cerevisiae expresses one member of the metacaspase Cys protease family, encoded by the YCA1 gene. Combination of proteomics and metabolomics data showed that YCA1 deletion down-regulated glycolysis, the TCA cycle and alcoholic fermentation as compared with WT cells. Δyca1 cells also showed a down-regulation of the pentose phosphate pathway and accumulation of pyruvate, correlated with higher levels of certain amino acids found in these cells. Accordingly, there is a decrease in protein biosynthesis, and up-regulation of specific stress response proteins like Ahp1p, which possibly provides these cells with a better protection against stress. Moreover, in agreement with the down-regulation of protein biosynthesis machinery in Δyca1 cells, we have found that regulation of transcription, co-translational protein folding and protein targeting to different subcellular locations were also down-regulated. Metabolomics analysis of the nucleotide content showed a significant reduction in Δyca1 cells in comparison with the WT, except for GTP content which remained unchanged. Thus, our combined proteome-metabolome approach added a new dimension to the non-apoptotic function of yeast metacaspase, which can specifically affect cell metabolism through as yet unknown mechanisms and possibly stress-response pathways, like HOG and cell wall integrity pathways. Certainly, YCA1 deletion may induce compensatory changes in stress response proteins offering a better protection against apoptosis to Δyca1 cells rather than a loss in pro-apoptotic YCA1-associated activity.

  14. Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus).

    PubMed

    Kim, Min Sun; Kim, Ki Hong

    2012-03-01

    To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.

  15. Ferulic Acid Orchestrates Anti-Oxidative Properties of Danggui Buxue Tang, an Ancient Herbal Decoction: Elucidation by Chemical Knock-Out Approach

    PubMed Central

    Gong, Amy G. W.; Huang, Vincent Y.; Wang, Huai Y.; Lin, Huang Q.; Dong, Tina T. X.; Tsim, Karl W. K.

    2016-01-01

    Ferulic acid, a phenolic acid derived mainly from a Chinese herb Angelica Sinensis Radix (ASR), was reported to reduce the formation of free radicals. Danggui Buxue Tang (DBT), a herbal decoction composing of Astragali Radix (AR) and ASR, has been utilized for more than 800 years in China having known anti-oxidative property. Ferulic acid is a major active ingredient in DBT; however, the role of ferulic acid within the herbal mixture has not been resolved. In order to elucidate the function of ferulic acid within this herbal decoction, a ferulic acid-depleted herbal decoction was created and named as DBTΔfa. The anti-oxidative properties of chemically modified DBT decoction were systemically compared in cultured H9C2 rat cardiomyoblast cell line. The application of DBT and DBTΔfa into the cultures showed functions in (i) decreasing the reactive oxygen species (ROS) formation, detected by laser confocal; (ii) increasing of the activation of Akt; (iii) increasing the transcriptional activity of anti-oxidant response element (ARE); and (iv) increasing the expressions of anti-oxidant enzymes, i.e. NQO1 and GCLM. In all scenario, the aforementioned anti-oxidative properties of DBTΔfa in H9C2 cells were significantly reduced, as compared to authentic DBT. Thus, ferulic acid could be an indispensable chemical in DBT to orchestrate multi-components of DBT as to achieve maximal anti-oxidative functions. PMID:27824860

  16. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

    PubMed

    Finka, Andrija; Saidi, Younousse; Goloubinoff, Pierre; Neuhaus, Jean-Marc; Zrÿd, Jean-Pierre; Schaefer, Didier G

    2008-10-01

    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

  17. Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications for As Detoxification and Accumulation in Plants

    PubMed Central

    Liu, Wenju; Schat, Henk; Bliek, Mathijs; Chen, Yi; McGrath, Steve P.; George, Graham; Salt, David E.; Zhao, Fang-Jie

    2012-01-01

    Many plant species are able to reduce arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast. PMID:22879969

  18. Metabolic consequences of knocking out UGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor (L. Moench).

    PubMed

    Blomstedt, Cecilia K; O'Donnell, Natalie H; Bjarnholt, Nanna; Neale, Alan D; Hamill, John D; Møller, Birger Lindberg; Gleadow, Roslyn M

    2016-02-01

    Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

  19. Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear.

    PubMed

    Porton, B; Rodriguiz, R M; Phillips, L E; Gilbert, J W; Feng, J; Greengard, P; Kao, H-T; Wetsel, W C

    2010-04-01

    Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.

  20. Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear

    PubMed Central

    Porton, Barbara; Rodriguiz, Ramona M.; Phillips, Lindsey E.; Gilbert, John W.; Feng, Jian; Greengard, Paul; Kao, Hung-Teh; Wetsel, William C.

    2010-01-01

    Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb, and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 hr and 10 days after training and in social transmission of food preference at 20 min and 24 hr. Additionally, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 hr after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. Since synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders. PMID:20050925

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

    PubMed

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

    2013-05-01

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

  2. miR-34a knockout attenuates cognitive deficits in APP/PS1 mice through inhibition of the amyloidogenic processing of APP.

    PubMed

    Jian, Chongdong; Lu, Mengru; Zhang, Zhao; Liu, Long; Li, Xianfeng; Huang, Fang; Xu, Ning; Qin, Lina; Zhang, Qian; Zou, Donghua

    2017-08-01

    The noncoding miRNA-34a (miR-34a) is involved in Alzheimer's disease (AD) pathologenesis and shows potential for application as a biomarker for early diagnosis and intervention. Here, we established miR-34a knockout mice in an APP/PS1 background (APP/PS1-miR-34a KO mice) by crossbreeding miR-34a(-/-) mice with APP/PS1 mice. We then investigated cognitive impairment and related pathologies. The results showed that the level of miR-34a was increased at about 6months in APP/PS1 mice, consistent with the increase in amyloid β (Aβ), and cognitive function was significantly improved in mice when miR-34a was knocked out in 9-month-old and 12-month-old mice, indicating that miR-34a is a potential candidate for determining the progression of AD. Furthermore, we assessed the processing of amyloid precursor protein (APP) and the results suggest that cognitive improvement by miR-34a knock out was mainly triggered by depression of γ-secretase activity, without affecting β- and α-secretase activities, indicating that miR-34a plays an important role in AD pathology, mainly by inhibiting the amyloidogenic processing of APP, without altering the non-amyloidogenic processing of APP. Copyright © 2017. Published by Elsevier Inc.

  3. Aged mice receiving caffeine since adulthood show distinct patterns of anxiety-related behavior.

    PubMed

    Botton, Paulo Henrique S; Pochmann, Daniela; Rocha, Andreia S; Nunes, Fernanda; Almeida, Amanda S; Marques, Daniela M; Porciúncula, Lisiane O

    2017-03-01

    Caffeine is the psychostimulant most consumed worldwide. Anxiogenic effects of caffeine have been described in adult animals with controversial findings about its anxiogenic potential. Besides, the effects of caffeine on anxiety with aging are still poorly known. In this study, adult mice (6months old) started to receive caffeine (0.3 and 1.0mg/mL, drinking water) during 12-14months only in the light cycle and at weekdays. The open field (OF) and elevated plus maze (EPM) testing were used to determine the effects of caffeine on anxiety-related behavior in adult and aged mice (18-20months old). Because aging alters synaptic proteins, we also evaluated SNAP-25 (as a nerve terminals marker), GFAP (as an astrocyte marker) and adenosine A1 and A2A receptors levels in the cortex. According to the OF analysis, caffeine did not change both hypolocomotion and anxiety with aging. However, aged mice showed less anxiety behavior in the EPM, but after receiving caffeine (0.3mg/mL) during adulthood they were anxious as adult mice. While SNAP-25 and adenosine A2A receptors increased with aging, both GFAP and adenosine A1 receptors were not affected. Caffeine at moderate dose prevented the age-related increase of the SNAP-25, with no effect on adenosine A2A receptors. The absence of effect for the highest dose suggests that tolerance to caffeine may have developed over time. Aged mice showed high responsiveness to the OF, being difficult to achieve any effect of caffeine. On the other hand this substance sustained the adult anxious behavior over time in a less stressful paradigm, and this effect was coincident with changes in the SNAP-25, suggesting the involvement of this synaptic protein in the ability of caffeine to preserve changes related to emotionality with aging.

  4. Transgenes and knock-outs in autoimmunity.

    PubMed

    Benoist, C; Mathis, D

    1993-12-01

    While there have not been any earth-shattering events during the past year relating to the use of germline manipulation in the study of autoimmunity, several new developments have brought interesting insights into the way the immune system deals (or fails to deal) with autoantigens. Several systems described recently have the potential to help us understand what makes an autoantigen, and what events lead to a pathogenic reaction.

  5. Juvenile mice show greater flexibility in multiple choice reversal learning than adults

    PubMed Central

    Johnson, Carolyn; Wilbrecht, Linda

    2011-01-01

    We hypothesized that decision-making strategies in juvenile animals, rather than being immature, are optimized to navigate the uncertainty and instability likely to be encountered in the environment at the time of the animal’s transition to independence. We tested juvenile and young adult mice on discrimination and reversal of a 4-choice and 2-choice odor-based foraging task. Juvenile mice (P26–27) learned a 4-choice discrimination and reversal faster than adults (P60–70), making fewer perseverative and distraction errors. Juvenile mice had shorter choice latencies and more focused search strategies. In both ages, performance of the task was significantly impaired by a lesion of the dorsomedial frontal cortex. Our data show that the frontal cortex can support highly flexible behavior in juvenile mice at a time coincident with weaning and first independence. The unexpected developmental decline in flexibility of behavior one month later suggests that frontal cortex based executive function may not inevitably become more flexible with age, but rather may be developmentally tuned to optimize exploratory and exploitative behavior for each life stage. PMID:21949556

  6. Mice deficient in dihydrolipoyl succinyltransferase show increased vulnerability to mitochondrial toxins

    PubMed Central

    Yang, Lichuan; Shi, Qingli; Ho, Daniel J.; Starkov, Anatoly A.; Wille, Elizabeth J.; Xu, Hui; Chen, HL; Zhang, Steven; Stack, Cliona M.; Calingasan, Noel Y.; Gibson, Gary E.; Beal, M. Flint

    2013-01-01

    The activity of a key mitochondrial tricarboxylic acid cycle enzyme, α-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyltransferase (DLST) component is unique to KGDHC. DLST+/- mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST+/- mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST+/- mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST+/- mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases. PMID:19660549

  7. Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins.

    PubMed

    Yang, Lichuan; Shi, Qingli; Ho, Daniel J; Starkov, Anatoly A; Wille, Elizabeth J; Xu, Hui; Chen, H L; Zhang, Steven; Stack, Cliona M; Calingasan, Noel Y; Gibson, Gary E; Beal, M Flint

    2009-11-01

    The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

  8. Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.

    PubMed

    Chidgey, M; Brakebusch, C; Gustafsson, E; Cruchley, A; Hail, C; Kirk, S; Merritt, A; North, A; Tselepis, C; Hewitt, J; Byrne, C; Fassler, R; Garrod, D

    2001-11-26

    The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.

  9. Granzyme K-deficient mice show no evidence of impaired antiviral immunity.

    PubMed

    Joeckel, Lars T; Allison, Cody C; Pellegrini, Marc; Bird, Catherina H; Bird, Phillip I

    2017-04-21

    The biological role of granzyme K, a serine protease of cytotoxic T lymphocytes (CTL), is controversial. It has been reported to induce perforin-mediated cell death in vitro, but is also reported to be non-cytotoxic and to operate in inflammatory processes. To elucidate the biological role of this protease, we have deleted the granzyme K gene in mice (mutant allele: Gzmk(tm1.1Pib); MGI:5636646). Gzmk (-/-) mice are healthy, anatomically normal, fecund and show normal hematopoietic development. Gzmk (-/-) mice readily recover from lymphocytic choriomeningitis virus and mouse pox Ectromelia virus infection. Ex vivo, virus-specific granzyme K-deficient CTL are indistinguishable from those of wild-type mice in apoptosis induction of target cells. These data suggest that granzyme K does not play an essential role in viral immunity or cytotoxicity. Our granzyme K knockout line completes the collection of mouse models for the human granzymes, and will further our understanding of their biological roles and relationships.Immunology and Cell Biology advance online publication, 23 May 2017; doi:10.1038/icb.2017.35.

  10. Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation

    PubMed Central

    Chidgey, Martyn; Brakebusch, Cord; Gustafsson, Erika; Cruchley, Alan; Hail, Chris; Kirk, Sarah; Merritt, Anita; North, Alison; Tselepis, Chris; Hewitt, Jane; Byrne, Carolyn; Fassler, Reinhard; Garrod, David

    2001-01-01

    The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation. PMID:11714727

  11. Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

    PubMed

    Kouno, Tetsuya; Akiyama, Nobuteru; Ito, Takahito; Okuda, Tomohiko; Nanchi, Isamu; Notoya, Mitsuru; Oka, Shogo; Yukioka, Hideo

    2016-02-01

    Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food. © 2016 Society for Endocrinology.

  12. Aged PrP null mice show defective processing of neuregulins in the peripheral nervous system.

    PubMed

    Benvegnù, Stefano; Gasperini, Lisa; Legname, Giuseppe

    2011-05-01

    A prion, a protease-resistant conformer of the cellular prion protein (PrP(C)), is the causative agent of transmissible spongiform encephalopathies or prion diseases. While this property is well established for the aberrantly folded protein, the physiological function of PrP(C) remains elusive. Among different putative functions, the non-pathogenic protein isoform PrP(C) is involved in several cellular processes. Here, we show that PrP(C) regulates the cleavage of neuregulin-1 proteins (NRG1). Neuregulins provide key axonal signals that regulate several processes, including glial cells proliferation, survival and myelination. Interestingly, mice devoid of PrP(C) (Prnp⁰/⁰) were recently shown to have a late-onset demyelinating disease in the peripheral nervous system (PNS) but not in the central nervous system (CNS). We found that NRG1 processing is developmentally regulated in the PNS and, by comparing wildtype and Prnp⁰/⁰ mice, that PrP(C) influences NRG1 processing in old, but not in young, animals. In addition, we found that also the processing of neuregulin-3, another neuregulin family member, is altered in the PNS of Prnp⁰/⁰ mice. These differences in neuregulin proteins processing are not paralleled in the CNS, thus suggesting a different cellular function for PrP(C) between the CNS and the PNS.

  13. Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice.

    PubMed

    Changklungmoa, Narin; Kueakhai, Pornanan; Riengrojpitak, Suda; Chaithirayanon, Kulathida; Chaichanasak, Pannigan; Preyavichyapugdee, Narin; Chantree, Pathanin; Sansri, Veerawat; Itagaki, Tadashi; Sobhon, Prasert

    2013-10-01

    Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 μg of rFgLAP combined with Freund's adjuvant (n = 10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3%, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.

  14. Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation.

    PubMed

    Kim, Gisen; Turovskaya, Olga; Levin, Matthew; Byrne, Fergus R; Whoriskey, John S; McCabe, James G; Kronenberg, Mitchell

    2008-10-15

    The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.

  15. Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation1

    PubMed Central

    Kim, Gisen; Turovskaya, Olga; Levin, Matthew; Byrne, Fergus R.; Whoriskey, John S.; McCabe, James G.; Kronenberg, Mitchell

    2009-01-01

    The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4+ T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3+ regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases. PMID:18832683

  16. Inbred mice strain shows neurobehavioral changes when exposed to tannery effluent.

    PubMed

    de Souza, Joyce Moreira; da Silva, Wellington Alves Mizael; de Oliveira Mendes, Bruna; Guimarães, Abraão Tiago Batista; de Lima Rodrigues, Aline Sueli; Montalvão, Mateus Flores; da Costa Estrela, Dieferson; da Silva, Anderson Rodrigo; Malafaia, Guilherme

    2017-01-01

    The bovine leather processing (tanning industries) stands as a generating activity of potentially toxic waste. The emission of untreated effluents into the environment may cause serious harm to human and environmental health. Nevertheless, few studies have investigated the possible effects of intake of these effluents in experimental mammalian models. Thus, this study aimed to evaluate the neurobehavioral effects of chronic intake of different tannery effluent concentrations diluted with water (0.1, 1, and 5%) in male C57BL/6J mice. After 120 days of exposure, the animals were subjected to different behavioral tests, predictive of anxiety (elevated plus maze (EPM), open-field (OF), and neophobia test), depression (forced swim), and memory deficits (object recognition test). From the EPM test, it was observed that the mice exposed to 0.1, 1, and 5% of tannery effluents showed higher anxiety scores compared to the animals in the control group. However, the results of this study revealed no differences among the experimental groups in the proportion (percentage) of locomotion in the central quarters/total locomotion calculated (by OF), considered an indirect measure for anxiety. At neophobia test, all the animals exposed to chronic intake of tannery effluents showed higher latency time to start eating, which corresponds to an anxiogenic behavior. Regarding the forced swim test, it was observed that the animals exposed to tannery effluents had longer time in immobility behavior, suggesting a predictive behavior to depression. Finally, the object recognition test showed that the treatments did not cause damage to the animals' memory. The recognition rate of the new object did not differ among the experimental groups. Thus, it is concluded that male C57BL/6J mice (inbred strain) exposed to tannery effluents have predictive neurobehavioral changes of anxiety and depression, without memory deficit.

  17. Fractures in geriatric mice show decreased callus expansion and bone volume.

    PubMed

    Lopas, Luke A; Belkin, Nicole S; Mutyaba, Patricia L; Gray, Chancellor F; Hankenson, Kurt D; Ahn, Jaimo

    2014-11-01

    210.79±37.60 mg HA/cm3; p=0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279±46,755 square pixels versus 302,167±137,806 square pixels; p=0.013) and bone content (mean bone volume at 20 days postfracture, 11.68±3.18 mm3 versus 22.34±10.59 mm3; p<0.001) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48±0.10 versus 0.50±0.13; p=0.793), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135+25 versus 90±52; p=0.221) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2±5.3 versus 18.7±5.2; p=0.324) was similar. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25%±6.8% versus 42%±14.5%; p=0.047). Our findings suggest that the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude. Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.

  18. Mice lacking brain-type creatine kinase activity show defective thermoregulation

    PubMed Central

    Streijger, Femke; Pluk, Helma; Oerlemans, Frank; Beckers, Gaby; Bianco, Antonio C.; Ribeiro, Miriam O.; Wieringa, Bé; Van der Zee, Catharina E.E.M.

    2010-01-01

    The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0°C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK−−/−− mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK−−/−− mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect. PMID:19419668

  19. Two Genetically Similar H9N2 Influenza A Viruses Show Different Pathogenicity in Mice

    PubMed Central

    Liu, Qingtao; Liu, Yuzhuo; Yang, Jing; Huang, Xinmei; Han, Kaikai; Zhao, Dongmin; Bi, Keran; Li, Yin

    2016-01-01

    H9N2 Avian influenza virus has repeatedly infected humans and other mammals, which highlights the need to determine the pathogenicity and the corresponding mechanism of this virus for mammals. In this study, we found two H9N2 viruses with similar genetic background but with different pathogenicity in mice. The A/duck/Nanjing/06/2003 (NJ06) virus was highly pathogenic for mice, with a 50% mouse lethal dose (MLD50) of 102.83 50% egg infectious dose (EID50), whereas the A/duck/Nanjing/01/1999 (NJ01) virus was low pathogenic for mice, with a MLD50 of >106.81 EID50. Further studies showed that the NJ06 virus grew faster and reached significantly higher titers than NJ01 in vivo and in vitro. Moreover, the NJ06 virus induced more severe lung lesions, and higher levels of inflammatory cellular infiltration and cytokine response in lungs than NJ01 did. However, only 12 different amino acid residues (HA-K157E, NA-A9T, NA-R435K, PB2-T149P, PB2-K627E, PB1-R187K, PA-L548M, PA-M550L, NP-G127E, NP-P277H, NP-D340N, NS1-D171N) were found between the two viruses, and all these residues except for NA-R435K were located in the known functional regions involved in interaction of viral proteins or between the virus and host factors. Summary, our results suggest that multiple amino acid differences may be responsible for the higher pathogenicity of the NJ06 virus for mice, resulting in lethal infection, enhanced viral replication, severe lung lesions, and excessive inflammatory cellular infiltration and cytokine response in lungs. These observations will be helpful for better understanding the pathogenic potential and the corresponding molecular basis of H9N2 viruses that might pose threats to human health in the future. PMID:27867373

  20. MRL/MpJ-Fas(lpr) mice show abnormalities in ovarian function and morphology with the progression of autoimmune disease.

    PubMed

    Otani, Yuki; Ichii, Osamu; Otsuka-Kanazawa, Saori; Chihara, Masataka; Nakamura, Teppei; Kon, Yasuhiro

    2015-01-01

    The immune system is known to affect reproductive function, and maternal-fetal immune tolerance is essential for a successful pregnancy. To investigate the relationship between autoimmune disease and female reproductive function, we performed a comparative analysis of the ovarian phenotypes for C57BL/6 mice, autoimmune disease-prone MRL/MpJ (MRL/+) mice and congenic MRL/MpJ-Fas(lpr) (MRL/lpr) mice harboring a mutation in the Fas gene that speeds disease onset. Both MRL-background strains showed earlier vaginal opening than C57BL/6 mice. The estrous cycle became irregular by 6 and 12 months of age in MRL/lpr mice and mice of the other two strains, respectively. Histological analysis at 3 months revealed that the number of primordial follicles was smaller in MRL-background mice than in C57BL/6 mice after 3 months. In addition, MRL/lpr and MRL/+ mice displayed lower numbers of ovarian follicles and corpora lutea at 3 and 6 months, and 6 and 12 months, respectively, than that in age-matched C57BL/6 mice. MRL/lpr and MRL/+ mice developed ovarian interstitial glands after 3 and 6 months, respectively. In particular, MRL/lpr mice showed numerous infiltrating lymphocytes within the ovarian interstitia, and partially stratified ovarian surface epithelia with more developed microvilli than that observed in C57BL/6 mice at 6 months. No significant differences in serum hormone levels were observed between the strains. In conclusion, MRL/lpr mice display altered ovarian development, morphology and function consistent with the progression of severe autoimmune disease, as these findings are less severe in MRL/+ counterparts.

  1. Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells.

    PubMed

    Mufazalov, Ilgiz A; Regen, Tommy; Schelmbauer, Carsten; Kuschmann, Janina; Muratova, Alisa M; Nikolaev, Alexei; Müller, Werner; Pinteaux, Emmanuel; Waisman, Ari

    2016-01-01

    Interleukin-1 (IL-1) plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1). To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN)-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment.

  2. Activation of NADPH-recycling systems in leaves and roots of Arabidopsis thaliana under arsenic-induced stress conditions is accelerated by knock-out of Nudix hydrolase 19 (AtNUDX19) gene.

    PubMed

    Corpas, Francisco J; Aguayo-Trinidad, Simeón; Ogawa, Takahisa; Yoshimura, Kazuya; Shigeoka, Shigeru

    2016-03-15

    NADPH is an important cofactor in cell growth, proliferation and detoxification. Arabidopsis thaliana Nudix hydrolase 19 (AtNUDX19) belongs to a family of proteins defined by the conserved amino-acid sequence GX5-EX7REUXEEXGU which has the capacity to hydrolyze NADPH as a physiological substrate in vivo. Given the importance of NADPH in the cellular redox homeostasis of plants, the present study compares the responses of the main NADPH-recycling systems including NADP-isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and NADP-malic enzyme (ME) in the leaves and roots of Arabidopsis wild-type (Wt) and knock-out (KO) AtNUDX19 mutant (Atnudx19) plants under physiological and arsenic-induced stress conditions. Two major features were observed in the behavior of the main NADPH-recycling systems: (i) under optimal conditions in both organs, the levels of these activities were higher in nudx19 mutants than in Wt plants; and, (ii) under 500μM AsV conditions, these activities increase, especially in nudx19 mutant plants. Moreover, G6PDH activity in roots was the most affected enzyme in both Wt and nudx19 mutant plants, with a 4.6-fold and 5.0-fold increase, respectively. In summary, the data reveals a connection between the absence of chloroplastic AtNUDX19 and the rise in all NADP-dehydrogenase activities under physiological and arsenic-induced stress conditions, particularly in roots. This suggests that AtNUDX19 could be a key factor in modulating the NADPH pool in plants and consequently in redox homeostasis. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. "Binge" drinking experience in adolescent mice shows sex differences and elevated ethanol intake in adulthood.

    PubMed

    Strong, Moriah N; Yoneyama, Naomi; Fretwell, Andrea M; Snelling, Chris; Tanchuck, Michelle A; Finn, Deborah A

    2010-06-01

    Binge drinking, defined as achieving blood ethanol concentrations (BEC) of 80 mg%, has been increasing in adolescents and was reported to predispose later physical dependence. The present experiments utilized an animal model of binge drinking to compare the effect of ethanol "binge" experience during adolescence or adulthood on subsequent ethanol intake in male and female C57BL/6 mice. Adolescent and adult mice were initially exposed to the scheduled high alcohol consumption procedure, which produces BECs that exceed the levels for binge drinking following a 30-min ethanol session every third day. Ethanol intake and BECs were significantly higher in the adolescent ( approximately 3 g/kg, 199 mg%) versus adult ( approximately 2 g/kg, 135 mg%) mice during the first three ethanol sessions, but were more equivalent during the final two ethanol sessions (1.85-2.0 g/kg, 129-143 mg%). Then, separate groups of the ethanol-experienced mice were tested with ethanol naïve adolescent and adult mice for 2-h limited access (10% and 20% solutions) or 24-h (5%, 10% and 20% solutions) ethanol preference drinking. Limited access ethanol intake was significantly higher in female versus male mice, but was not altered by age or ethanol experience. In contrast, 24-h ethanol intake was significantly higher in the adolescent versus adult mice and in female versus male mice. Furthermore, binge drinking experience in the adolescent mice significantly increased subsequent ethanol intake, primarily due to intake in female mice. Thus, adolescent binge drinking significantly increased unlimited ethanol intake during adulthood, with female mice more susceptible to this effect.

  4. Ceruloplasmin gene-deficient mice with experimental autoimmune encephalomyelitis show attenuated early disease evolution.

    PubMed

    Gresle, Melissa M; Schulz, Katrin; Jonas, Anna; Perreau, Victoria M; Cipriani, Tania; Baxter, Alan G; Miranda-Hernandez, Socorro; Field, Judith; Jokubaitis, Vilija G; Cherny, Robert; Volitakis, Irene; David, Samuel; Kilpatrick, Trevor J; Butzkueven, Helmut

    2014-06-01

    We conducted a microarray study to identify genes that are differentially regulated in the spinal cords of mice with the inflammatory disease experimental autoimmune encephalomyelitis (EAE) relative to healthy mice. In total 181 genes with at least a two-fold increase in expression were identified, and most of these genes were associated with immune function. Unexpectedly, ceruloplasmin (Cp), a ferroxidase that converts toxic ferrous iron to its nontoxic ferric form and also promotes the efflux of iron from astrocytes in the CNS, was shown to be highly upregulated (13.2-fold increase) in EAE spinal cord. Expression of Cp protein is known to be increased in several neurological conditions, but the role of Cp regulation in CNS autoimmune disease is not known. To investigate this, we induced EAE in Cp gene knockout, heterozygous, and wild-type mice. Cp knockout mice were found to have slower disease evolution than wild-type mice (EAE days 13-17; P = 0.05). Interestingly, Cp knockout mice also exhibited a significant increase in the number of astrocytes with reactive morphology in early EAE compared with wild-type mice at the same stage of disease. CNS iron levels were not increased with EAE in these mice. Based on these observations, we propose that an increase in Cp expression could contribute to tissue damage in early EAE. In addition, endogenous CP either directly or indirectly inhibits astrocyte reactivity during early disease, which could also worsen early disease evolution.

  5. Hepatic Farnesoid X-Receptor Isoforms α2 and α4 Differentially Modulate Bile Salt and Lipoprotein Metabolism in Mice

    PubMed Central

    Boesjes, Marije; Bloks, Vincent W.; Hageman, Jurre; Bos, Trijnie; van Dijk, Theo H.; Havinga, Rick; Wolters, Henk; Jonker, Johan W.; Kuipers, Folkert; Groen, Albert K.

    2014-01-01

    The nuclear receptor FXR acts as an intracellular bile salt sensor that regulates synthesis and transport of bile salts within their enterohepatic circulation. In addition, FXR is involved in control of a variety of crucial metabolic pathways. Four FXR splice variants are known, i.e. FXRα1-4. Although these isoforms show differences in spatial and temporal expression patterns as well as in transcriptional activity, the physiological relevance hereof has remained elusive. We have evaluated specific roles of hepatic FXRα2 and FXRα4 by stably expressing these isoforms using liver-specific self-complementary adeno-associated viral vectors in total body FXR knock-out mice. The hepatic gene expression profile of the FXR knock-out mice was largely normalized by both isoforms. Yet, differential effects were also apparent; FXRα2 was more effective in reducing elevated HDL levels and transrepressed hepatic expression of Cyp8b1, the regulator of cholate synthesis. The latter coincided with a switch in hydrophobicity of the bile salt pool. Furthermore, FXRα2-transduction caused an increased neutral sterol excretion compared to FXRα4 without affecting intestinal cholesterol absorption. Our data show, for the first time, that hepatic FXRα2 and FXRα4 differentially modulate bile salt and lipoprotein metabolism in mice. PMID:25506828

  6. Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

    SciTech Connect

    Wang, Yuehai; Lu, Huixia; Huang, Ziyang; Lin, Huili; Lei, Zhenmin; Tang, Mengxiong; Gao, Fei; Dong, Mei; Li, Rongda; Lin, Ling

    2014-07-18

    Highlights: • Titers of ANA and anti-dsDNA antibodies were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • The spleen weights and glomerular areas were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • Expressions of IgG and C3 in glomeruli were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • IgG, C3 and macrophage infiltration in aortic plaques were found in ApoE{sup −/−} mice. - Abstract: Background: Apolipoprotein E-knockout (ApoE{sup −/−}) mice is a classic model of atherosclerosis. We have found that ApoE{sup −/−} mice showed splenomegaly, higher titers of serum anti-nuclear antibody (ANA) and anti-dsDNA antibody compared with C57B6/L (B6) mice. However, whether ApoE{sup −/−} mice show autoimmune injury remains unclear. Methods and results: Six females and six males in each group, ApoE{sup −/−}, Fas{sup −/−} and B6 mice, were used in this study. The titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein were measured by ELISA after 4 months of high-fat diet. The spleen weight and the glomerular area were determined. The expressions of IgG, C3 and macrophage in kidney and atherosclerotic plaque were detected by immunostaining followed by morphometric analysis. Similar to the characteristics of Fas{sup −/−} mice, a model of systemic lupus erythematosus (SLE), ApoE{sup −/−} mice, especially female, displayed significant increases of spleen weight and glomerular area when compared to B6 mice. Also, elevated titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein. Moreover, the expressions of IgG, C3 and macrophage in glomeruli and aortic plaques were found in ApoE{sup −/−} mice. In addition, the IgG and C3 expressions in glomeruli and plaques significantly increased (or a trend of increase) in female ApoE{sup −/−} mice compared with males. Conclusions: Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta.

  7. Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls

    PubMed Central

    Musolf, Kerstin; Meindl, Stefanie; Larsen, Angela L.; Kalcounis-Rueppell, Matina C.; Penn, Dustin J.

    2015-01-01

    Male house mice (Mus musculus) emit ultrasonic vocalizations (USVs) during courtship, which attract females, and we aimed to test whether females use these vocalizations for species or subspecies recognition of potential mates. We recorded courtship USVs of males from different Mus species, Mus musculus subspecies, and populations (F1 offspring of wild-caught Mus musculus musculus, Mus musculus domesticus (and F1 hybrid crosses), and Mus spicilegus), and we conducted playback experiments to measure female preferences for male USVs. Male vocalizations contained at least seven distinct syllable types, whose frequency of occurrence varied among species, subspecies, and populations. Detailed analyses of multiple common syllable types indicated that Mus musculus and Mus spicilegus could be discriminated based on spectral and temporal characteristics of their vocalizations, and populations of Mus musculus were also distinctive regardless of the classification model used. Females were able to discriminate USVs from different species, and showed assortative preferences for conspecific males. We found no evidence that females discriminate USVs of males from a different subspecies or separate populations of the same species, even though our spectral analyses identified acoustic features that differ between species, subspecies, and populations of the same species. Our results provide the first comparison of USVs between Mus species or between Mus musculus subspecies, and the first evidence that male USVs potentially facilitate species recognition. PMID:26309246

  8. Long-Lived αMUPA Mice Show Attenuation of Cardiac Aging and Leptin-Dependent Cardioprotection.

    PubMed

    Levy, Esther; Kornowski, Ran; Gavrieli, Reut; Fratty, Ilana; Greenberg, Gabriel; Waldman, Maayan; Birk, Einat; Shainberg, Asher; Akirov, Amit; Miskin, Ruth; Hochhauser, Edith

    2015-01-01

    αMUPA transgenic mice spontaneously consume less food compared with their wild type (WT) ancestors due to endogenously increased levels of the satiety hormone leptin. αMUPA mice share many benefits with mice under caloric restriction (CR) including an extended life span. To understand mechanisms linked to cardiac aging, we explored the response of αMUPA hearts to ischemic conditions at the age of 6, 18, or 24 months. Mice were subjected to myocardial infarction (MI) in vivo and to ischemia/reperfusion ex vivo. Compared to WT mice, αMUPA showed functional and histological advantages under all experimental conditions. At 24 months, none of the WT mice survived the first ischemic day while αMUPA mice demonstrated 50% survival after 7 ischemic days. Leptin, an adipokine decreasing under CR, was consistently ~60% higher in αMUPA sera at baseline. Leptin levels gradually increased in both genotypes 24h post MI but were doubled in αMUPA. Pretreatment with leptin neutralizing antibodies or with inhibitors of leptin signaling (AG-490 and Wortmannin) abrogated the αMUPA benefits. The antibodies also reduced phosphorylation of the leptin signaling components STAT3 and AKT specifically in the αMUPA myocardium. αMUPA mice did not show elevation in adiponectin, an adipokine previously implicated in CR-induced cardioprotection. WT mice treated for short-term CR exhibited cardioprotection similar to that of αMUPA, however, along with increased adiponectin at baseline. Collectively, the results demonstrate a life-long increased ischemic tolerance in αMUPA mice, indicating the attenuation of cardiac aging. αMUPA cardioprotection is mediated through endogenous leptin, suggesting a protective pathway distinct from that elicited under CR.

  9. Long-Lived αMUPA Mice Show Attenuation of Cardiac Aging and Leptin-Dependent Cardioprotection

    PubMed Central

    Levy, Esther; Kornowski, Ran; Gavrieli, Reut; Fratty, Ilana; Greenberg, Gabriel; Waldman, Maayan; Birk, Einat; Shainberg, Asher; Akirov, Amit; Miskin, Ruth; Hochhauser, Edith

    2015-01-01

    αMUPA transgenic mice spontaneously consume less food compared with their wild type (WT) ancestors due to endogenously increased levels of the satiety hormone leptin. αMUPA mice share many benefits with mice under caloric restriction (CR) including an extended life span. To understand mechanisms linked to cardiac aging, we explored the response of αMUPA hearts to ischemic conditions at the age of 6, 18, or 24 months. Mice were subjected to myocardial infarction (MI) in vivo and to ischemia/reperfusion ex vivo. Compared to WT mice, αMUPA showed functional and histological advantages under all experimental conditions. At 24 months, none of the WT mice survived the first ischemic day while αMUPA mice demonstrated 50% survival after 7 ischemic days. Leptin, an adipokine decreasing under CR, was consistently ~60% higher in αMUPA sera at baseline. Leptin levels gradually increased in both genotypes 24h post MI but were doubled in αMUPA. Pretreatment with leptin neutralizing antibodies or with inhibitors of leptin signaling (AG-490 and Wortmannin) abrogated the αMUPA benefits. The antibodies also reduced phosphorylation of the leptin signaling components STAT3 and AKT specifically in the αMUPA myocardium. αMUPA mice did not show elevation in adiponectin, an adipokine previously implicated in CR-induced cardioprotection. WT mice treated for short-term CR exhibited cardioprotection similar to that of αMUPA, however, along with increased adiponectin at baseline. Collectively, the results demonstrate a life-long increased ischemic tolerance in αMUPA mice, indicating the attenuation of cardiac aging. αMUPA cardioprotection is mediated through endogenous leptin, suggesting a protective pathway distinct from that elicited under CR. PMID:26673217

  10. Astrocytic leptin-receptor knockout mice show partial rescue of leptin resistance in diet-induced obesity.

    PubMed

    Jayaram, Bhavaani; Pan, Weihong; Wang, Yuping; Hsuchou, Hung; Mace, Aurelien; Cornelissen-Guillaume, Germaine G; Mishra, Pramod K; Koza, Robert A; Kastin, Abba J

    2013-03-15

    To determine how astrocytic leptin signaling regulates the physiological response of mice to diet-induced obesity (DIO), we performed metabolic analyses and hypothalamic leptin signaling assays on astrocytic leptin-receptor knockout (ALKO) mice in which astrocytes lack functional leptin receptor (ObR) signaling. ALKO mice and wild-type (WT) littermate controls were studied at different stages of DIO with measurement of body wt, percent fat, metabolic activity, and biochemical parameters. When fed regular chow, the ALKO mice had similar body wt, percent fat, food intake, heat dissipation, respiratory exchange ratio, and activity as their WT littermates. There was no change in blood concentrations of triglyceride, soluble leptin receptor (sObR), mRNA for leptin and uncoupling protein 1 (UCP1) in adipose tissue, and insulin sensitivity. Unexpectedly, in response to a high-fat diet the ALKO mice had attenuated hyperleptinemia and sObR, a lower level of leptin mRNA in subcutaneous fat, and a paradoxical increase in UCP1 mRNA. Thus, ALKO mice did not show the worsening of obesity that occurs with normal WT mice and the neuronal ObR mutation that results in morbid obesity. The findings are consistent with a competing, counterregulatory model between neuronal and astrocytic leptin signaling.

  11. 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.

  12. Young MLP deficient mice show diastolic dysfunction before the onset of dilated cardiomyopathy.

    PubMed

    Lorenzen-Schmidt, Ilka; Stuyvers, Bruno D; ter Keurs, Henk E D J; Date, Moto-o; Hoshijima, Masahiko; Chien, Kenneth R; McCulloch, Andrew D; Omens, Jeffrey H

    2005-08-01

    Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 microm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.

  13. Young MLP deficient mice show diastolic dysfunction before the onset of dilated cardiomyopathy

    PubMed Central

    Lorenzen-Schmidt, Ilka; Stuyvers, Bruno D.; ter Keurs, Henk E.D.J.; Date, Moto-o; Hoshijima, Masahiko; Chien, Kenneth R.; McCulloch, Andrew D.; Omens, Jeffrey H.

    2015-01-01

    Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 μm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction. PMID:15978612

  14. Polysaccharides from Laminaria japonica show hypoglycemic and hypolipidemic activities in mice with experimentally induced diabetes.

    PubMed

    Jia, Xibei; Yang, Juan; Wang, Zhi; Liu, Ruichan; Xie, Rujuan

    2014-12-01

    Diabetes mellitus (DM) is a chronic metabolic disorder of the endocrine system. The rapid increase in the incidence of DM is a serious public health concern worldwide. The treatment of DM and its complications mainly involves the use of chemically or biochemically synthesized drugs, but these drugs also have adverse side effects. Therefore, there is an urgent need to search for drugs from natural sources that would cause fewer side effects. This study aimed to determine whether polysaccharides from Laminaria japonica (LJP) exert hypoglycemic and hypolipidemic effects in mice with alloxan-induced diabetes. To this end, diabetes was induced by alloxan injection (200 mg/kg body weight [bw], intraperitoneal [ip]). After induction of diabetes, diabetic mice were randomly divided into five groups: diabetic control (DC) group, glibenclamide-treated (DG) group, low-dose LJP-treated (DLL) group, moderate-dose LJP-treated (DML) group, and high-dose LJP-treated (DHL) group, with normal mice used as the control group (NC group). After treatment for 28 days, body weight, fasting blood glucose (FBG), serum insulin, total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) levels were measured. The results revealed that LJP administration prevented body-weight loss, decreased FBG levels, and increased serum insulin levels in diabetic mice. Furthermore, it decreased TC, TG, and LDL-C levels, and increased HDL-C levels in these mice. Thus, the results indicate that LJP possesses hypoglycemic and hypolipidemic activities and polysaccharides from LJP may hold promise for the development of a drug of natural origin for the treatment of DM. © 2014 by the Society for Experimental Biology and Medicine.

  15. Apolipoprotein E-knockout mice show increased titers of serum anti-nuclear and anti-dsDNA antibodies

    SciTech Connect

    Wang, Yuehai; Huang, Ziyang; Lu, Huixia; Lin, Huili; Wang, Zhenhua; Chen, Xiaoqing; Ouyang, Qiufang; Tang, Mengxiong; Hao, Panpan; Ni, Jingqin; Xu, Dongming; Zhang, Mingxiang; Zhang, Qunye; Lin, Ling; and others

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Titers of ANA and anti-dsDNA antibodies were higher in ApoE{sup -/-} than C57B6/L mice. Black-Right-Pointing-Pointer Spleen was greater and splenocyte apoptosis lower in ApoE{sup -/-} than B6 mice. Black-Right-Pointing-Pointer Level of TLR4 was lower in spleen tissue of ApoE{sup -/-} than B6 mice. Black-Right-Pointing-Pointer The TLR4 pathway may participate in maintaining the balance of splenocyte apoptosis. Black-Right-Pointing-Pointer The TLR4 pathway may participate in antibody production in spleen tissue. -- Abstract: Apolipoprotein E-knockout (ApoE{sup -/-}) mice, atherosclerosis-prone mice, show an autoimmune response, but the pathogenesis is not fully understood. We investigated the pathogenesis in female and male ApoE{sup -/-} mice. The spleens of all ApoE{sup -/-} and C57BL/6 (B6) mice were weighed. The serum IgG level and titers of anti-nuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA) antibody were assayed by ELISA. Apoptosis of spleen tissue was evaluated by TUNEL. TLR4 level in spleen tissue was tested by immunohistochemistry and Western blot analysis. Levels of MyD88, p38, phosphorylated p38 (pp38), interferon regulatory factor 3 (IRF3) and Bcl-2-associated X protein (Bax) in spleen tissue were detected by Western blot analysis. We also survey the changes of serum autoantibodies, spleen weight, splenocyte apoptosis and the expressions of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue in male ApoE{sup -/-} mice after 4 weeks of lipopolysaccharide (LPS), Toll-like receptor 4 ligand, administration. ApoE{sup -/-} mice showed splenomegaly and significantly increased serum level of IgG and titers of ANA and anti-dsDNA antibody as compared with B6 mice. Splenocyte apoptosis and the expression of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue were significantly lower in ApoE{sup -/-} than B6 mice. The expression of TLR4, MyD88, IRF3, pp38, and Bax differed by sex in ApoE{sup -/-} spleen tissue. The

  16. Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced Fucalpha1-2 glycosyltransferase.

    PubMed Central

    Thomsson, Kristina A; Hinojosa-Kurtzberg, Marina; Axelsson, Karin A; Domino, Steven E; Lowe, John B; Gendler, Sandra J; Hansson, Gunnar C

    2002-01-01

    In gene-targeted mouse models for cystic fibrosis (CF), the disease is mainly manifested by mucus obstruction in the intestine. To explore the mucus composition, mucins insoluble and soluble in 6 M guanidinium chloride were purified by three rounds of isopycnic ultracentrifugation from the small and large intestines of CF mice (Cftr(m1UNC)/Cftr(m1UNC)) and compared with wild-type mice. The amino acid composition was typical of that for mucins and showed increased amounts of the insoluble (2.5-fold increase) and soluble (7-fold increase) mucins in the small intestine of the CF mice compared with wild-type mice. Mucins from the large intestine of both wild-type and CF mice showed a high but constant level of fucosylation. In contrast, the insoluble and soluble mucins of the small intestine in CF mice revealed a large increase in fucose, whereas those of wild-type mice contained only small amounts of fucose. This increased fucosylation was analysed by releasing the O-linked oligosaccharides followed by GC-MS. NMR spectroscopy revealed that the increased fucosylation was due to an increased expression of blood group H epitopes (Fucalpha1-2Gal-). Northern-blot analysis, using a probe for the murine Fucalpha1-2 fucosyltransferase (Fut2), showed an up-regulation of this mRNA in the small intestine of the CF mice, suggesting that this enzyme is responsible for the observed increase in blood group H-type glycosylation. The reason for this up-regulation could be a direct or indirect effect of a non-functional CF transmembrane conductance regulator (CFTR) caused by the absence of CFTR channel. PMID:12164788

  17. PACAP-deficient mice show attenuated corticosterone secretion and fail to develop depressive behavior during chronic social defeat stress.

    PubMed

    Lehmann, Michael L; Mustafa, Tomris; Eiden, Adrian M; Herkenham, Miles; Eiden, Lee E

    2013-05-01

    The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis and the adrenal gland in response to various stressors. We previously found that in response to acute psychological stress (restraint), elevated corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamic paraventricular nucleus (PVN) as well as elevated plasma corticosterone (CORT) were profoundly attenuated in PACAP-deficient mice. To determine whether HPA axis responses and stress-induced depressive-like behaviors in a chronic stress paradigm are affected by PACAP deficiency, we subjected mice to 14 days of social defeat stress. Defeat-exposed PACAP-/- mice showed a marked attenuation of stress-induced increases in serum CORT levels, cellular PVN ΔFosB immunostaining, and depressive-like behaviors (social interaction and forced swim tests) compared to wild-type control mice. The PACAP-/- mice showed reduced PVN FosB-positive cell numbers, but relatively elevated cell counts in several forebrain areas including the medial prefrontal cortex, after social stress. PACAP appears to be specific for mediating HPA activation only in psychological stress because marked elevations in plasma CORT after a systemic stressor (lipopolysaccharide administration) occurred regardless of genotype. We conclude that chronically elevated CORT is a key component of depressive effects of social defeat, and that attenuation of the CORT response at the level of the PVN, as well as extrahypothalamic forebrain regions, in PACAP-deficient mice protects from development of depressive behavior.

  18. Caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection.

    PubMed

    Medina, Freddy A; de Almeida, Cecilia J; Dew, Elliott; Li, Jiangwei; Bonuccelli, Gloria; Williams, Terence M; Cohen, Alex W; Pestell, Richard G; Frank, Philippe G; Tanowitz, Herbert B; Lisanti, Michael P

    2006-12-01

    A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1(-/-) mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1(-/-) mice. However, infection of Cav-1(-/-) macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1(-/-) mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1(-/-) mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1(-/-) mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1(-/-) macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

  19. Mice lacking hypertension candidate gene ATP2B1 in vascular smooth muscle cells show significant blood pressure elevation.

    PubMed

    Kobayashi, Yusuke; Hirawa, Nobuhito; Tabara, Yasuharu; Muraoka, Hidenori; Fujita, Megumi; Miyazaki, Nobuko; Fujiwara, Akira; Ichikawa, Yasuhiro; Yamamoto, Yuichiro; Ichihara, Naoaki; Saka, Sanae; Wakui, Hiromichi; Yoshida, Shin-ichiro; Yatsu, Keisuke; Toya, Yoshiyuki; Yasuda, Gen; Kohara, Katsuhiko; Kita, Yoshikuni; Takei, Kohtaro; Goshima, Yoshio; Ishikawa, Yoshihiro; Ueshima, Hirotsugu; Miki, Tetsuro; Umemura, Satoshi

    2012-04-01

    We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na(+)-Ca(2+) exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

  20. Sulfobetaine (dimethylsulfoniopropionate) and glycine betaine show incompatible involvement in crucial Ehrlich ascites carcinoma in mice.

    PubMed

    Nakajima, Kenji; Nakajima, Yoshiki; Tsujiwaki, Satomi

    2015-03-01

    The role of methylation reactions in cancer was examined using the methylating agents, sulfobetaine [dimethylsulfonioproponate (DMSP)], and glycine betaine (GB), in murine crucial Ehrlich ascites carcinoma (EAC) for up to 10 days. DMSP administration in EAC-bearing mice mitigated EAC, while GB administration clearly promoted EAC. However, the immune cell profiles did not differ largely between animals receiving DMSP and those receiving GB. Moreover, DMSP and GB had merely any effects on proliferation of EAC cells in vitro. Injection of DMSP into normal mice interestingly led to macrophage accumulation in the peritoneal cavity in a dose-dependent manner at early rearing. These results indicate that GB promoted EAC by the methylation of cancer promotor gene, whereas DMSP ameliorated EAC by the accumulation of activated macrophages with a rapid response and long life span during cancer progression. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  1. Female mice lacking Xist RNA show partial dosage compensation and survive to term

    PubMed Central

    Yang, Lin; Kirby, James E.; Sunwoo, Hongjae; Lee, Jeannie T.

    2016-01-01

    X-chromosome inactivation (XCI) compensates for differences in X-chromosome number between male and female mammals. XCI is orchestrated by Xist RNA, whose expression in early development leads to transcriptional silencing of one X chromosome in the female. Knockout studies have established a requirement for Xist with inviability of female embryos that inherit an Xist deletion from the father. Here, we report that female mice lacking Xist RNA can, surprisingly, develop and survive to term. Xist-null females are born at lower frequency and are smaller at birth, but organogenesis is mostly normal. Transcriptomic analysis indicates significant overexpression of hundreds of X-linked genes across multiple tissues. Therefore, Xist-null mice can develop to term in spite of a deficiency of dosage compensation. However, the degree of X-autosomal dosage imbalance was less than anticipated (1.14-fold to 1.36-fold). Thus, partial dosage compensation can be achieved without Xist, supporting the idea of inherent genome balance. Nevertheless, to date, none of the mutant mice has survived beyond weaning stage. Sudden death is associated with failure of postnatal organ maturation. Our data suggest Xist-independent mechanisms of dosage compensation and demonstrate that small deviations from X-autosomal balance can have profound effects on overall fitness. PMID:27542829

  2. Behavioral characterization of cereblon forebrain-specific conditional null mice: a model for human non-syndromic intellectual disability.

    PubMed

    Rajadhyaksha, Anjali M; Ra, Stephen; Kishinevsky, Sarah; Lee, Anni S; Romanienko, Peter; DuBoff, Mariel; Yang, Chingwen; Zupan, Bojana; Byrne, Maureen; Daruwalla, Zeeba R; Mark, Willie; Kosofsky, Barry E; Toth, Miklos; Higgins, Joseph J

    2012-01-15

    A nonsense mutation in the human cereblon gene (CRBN) causes a mild type of autosomal recessive non-syndromic intellectual disability (ID). Animal studies show that crbn is a cytosolic protein with abundant expression in the hippocampus (HPC) and neocortex (CTX). Its diverse functions include the developmental regulation of ion channels at the neuronal synapse, the mediation of developmental programs by ubiquitination, and a target for herpes simplex type I virus in HPC neurons. To test the hypothesis that anomalous CRBN expression leads to HPC-mediated memory and learning deficits, we generated germ-line crbn knock-out mice (crbn(-/-)). We also inactivated crbn in forebrain neurons in conditional knock-out mice in which crbn exons 3 and 4 are deleted by cre recombinase under the direction of the Ca(2+)/calmodulin-dependent protein kinase II alpha promoter (CamKII(cre/+), crbn(-/-)). crbn mRNA levels were negligible in the HPC, CTX, and cerebellum (CRBM) of the crbn(-/-) mice. In contrast, crbn mRNA levels were reduced 3- to 4-fold in the HPC, CTX but not in the CRBM in CamKII(cre/+), crbn(-/-) mice as compared to wild type (CamKII(cre/+), crbn(+/+)). Contextual fear conditioning showed a significant decrease in the percentage of freezing time in CamKII(cre/+), crbn(-/-) and crbn(-/-) mice while motor function, exploratory motivation, and anxiety-related behaviors were normal. These findings suggest that CamKII(cre/+), crbn(-/-) mice exhibit selective HPC-dependent deficits in associative learning and supports the use of these mice as in vivo models to study the functional consequences of CRBN aberrations on memory and learning in humans.

  3. IL1RAPL1 knockout mice show spine density decrease, learning deficiency, hyperactivity and reduced anxiety-like behaviours.

    PubMed

    Yasumura, Misato; Yoshida, Tomoyuki; Yamazaki, Maya; Abe, Manabu; Natsume, Rie; Kanno, Kouta; Uemura, Takeshi; Takao, Keizo; Sakimura, Kenji; Kikusui, Takefumi; Miyakawa, Tsuyoshi; Mishina, Masayoshi

    2014-10-14

    IL-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic intellectual disability and is associated with autism. IL1RAPL1 mediates excitatory synapse formation through trans-synaptic interaction with PTPδ. Here, we showed that the spine density of cortical neurons was significantly reduced in IL1RAPL1 knockout mice. The spatial reference and working memories and remote fear memory were mildly impaired in IL1RAPL1 knockout mice. Furthermore, the behavioural flexibility was slightly reduced in the T-maze test. Interestingly, the performance of IL1RAPL1 knockout mice in the rotarod test was significantly better than that of wild-type mice. Moreover, IL1RAPL1 knockout mice consistently exhibited high locomotor activity in all the tasks examined. In addition, open-space and height anxiety-like behaviours were decreased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 ablation resulted in spine density decrease and affected not only learning but also behavioural flexibility, locomotor activity and anxiety.

  4. IL1RAPL1 knockout mice show spine density decrease, learning deficiency, hyperactivity and reduced anxiety-like behaviours

    PubMed Central

    Yasumura, Misato; Yoshida, Tomoyuki; Yamazaki, Maya; Abe, Manabu; Natsume, Rie; Kanno, Kouta; Uemura, Takeshi; Takao, Keizo; Sakimura, Kenji; Kikusui, Takefumi; Miyakawa, Tsuyoshi; Mishina, Masayoshi

    2014-01-01

    IL-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic intellectual disability and is associated with autism. IL1RAPL1 mediates excitatory synapse formation through trans-synaptic interaction with PTPδ. Here, we showed that the spine density of cortical neurons was significantly reduced in IL1RAPL1 knockout mice. The spatial reference and working memories and remote fear memory were mildly impaired in IL1RAPL1 knockout mice. Furthermore, the behavioural flexibility was slightly reduced in the T-maze test. Interestingly, the performance of IL1RAPL1 knockout mice in the rotarod test was significantly better than that of wild-type mice. Moreover, IL1RAPL1 knockout mice consistently exhibited high locomotor activity in all the tasks examined. In addition, open-space and height anxiety-like behaviours were decreased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 ablation resulted in spine density decrease and affected not only learning but also behavioural flexibility, locomotor activity and anxiety. PMID:25312502

  5. Factor XIII-A transglutaminase deficient mice show signs of metabolically healthy obesity on high fat diet

    PubMed Central

    Myneni, Vamsee D.; Mousa, Aisha; Kaartinen, Mari T.

    2016-01-01

    F13A1 gene, which encodes for Factor XIII-A blood clotting factor and a transglutaminase enzyme, was recently identified as a potential causative gene for obesity in humans. In our previous in vitro work, we showed that FXIII-A regulates preadipocyte differentiation and modulates insulin signaling via promoting plasma fibronectin assembly into the extracellular matrix. To understand the role of FXIII-A in whole body energy metabolism, here we have characterized the metabolic phenotype of F13a1−/− mice. F13a1−/− and F13a1+/+ type mice were fed chow or obesogenic, high fat diet for 20 weeks. Weight gain, total fat mass and fat pad mass, glucose handling, insulin sensitivity, energy expenditure and, morphological and biochemical analysis of adipose tissue was performed. We show that mice lacking FXIII-A gain weight on obesogenic diet, similarly as wild type mice, but exhibit a number of features of metabolically healthy obesity such as protection from developing diet-induced insulin resistance and hyperinsulinemia. Mice also show normal fasting glucose levels, larger adipocytes, decreased extracellular matrix accumulation and inflammation of adipose tissue, as well as decreased circulating triglycerides. This study reveals that FXIII-A transglutaminase can regulate whole body insulin sensitivity and may have a role in the development of diet-induced metabolic disturbances. PMID:27759118

  6. ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease

    PubMed Central

    Komnig, Daniel; Imgrund, Silke; Reich, Arno; Gründer, Stefan; Falkenburger, Björn H.

    2016-01-01

    Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride. PMID:27820820

  7. ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease.

    PubMed

    Komnig, Daniel; Imgrund, Silke; Reich, Arno; Gründer, Stefan; Falkenburger, Björn H

    2016-01-01

    Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

  8. Central administration of obestatin fails to show inhibitory effects on food and water intake in mice.

    PubMed

    Van Dijck, Annemie; Annemie, Van Dijck; Van Dam, Debby; Debby, Van Dam; Vergote, Valentijn; Valentijn, Vergote; De Spiegeleer, Bart; Bart, De Spiegeleer; Luyten, Walter; Walter, Luyten; Schoofs, Liliane; Liliane, Schoofs; De Deyn, Peter Paul; Peter Paul, De Deyn

    2009-08-07

    Obestatin is a ghrelin-associated peptide hormone with presumed anorexigenic and inhibitory effect on gastric propulsive motility activity. Recent literature, however, discloses much contestation over satiety and gastrointestinal motility-related functionalities of obestatin. In addition, antidipsinogenic effects in rodents by obestatin were recently reported. The present study was set up to bring more clarity into the contested effects of obestatin on food and water intake. Additionally, the stability of obestatin in brain tissue homogenate was investigated. The in vitro incubation of obestatin in brain homogenates revealed disappearance half-life times of 19 min for crude brain homogenate to 27 min for brain membrane homogenate. For the behavioural studies, male C57Bl/6 mice were intracerebroventricularly treated with 0.2 nmol murine amidated obestatin or vehicle at the age of 3 months. An additional group of mice was treated with 0.3 nmol of corticotropin releasing factor (CRF) as a positive control of suppression of food intake. Food and water intake were studied over a period of 5 h in metabolic cages. Under our experimental conditions, no suppressive effects of obestatin on food or water intake were observed, whereas CRF evoked a significant suppression of food intake, which proves the internal validity of the study design.

  9. Reduced acute nociception and chronic pain in Shank2−/− mice

    PubMed Central

    Ko, Hyoung-Gon; Oh, Seog-Bae; Zhuo, Min

    2016-01-01

    Autism spectrum disorder is a debilitating mental illness and social issue. Autism spectrum disorder patients suffer from social isolation, cognitive deficits, compulsive behavior, and sensory deficits, including hyposensitivity to pain. However, recent studies argued that autism spectrum disorder patients show physiological pain response and, in some cases, even extremely intense pain response to harmless stimulation. Recently, Shank gene family was reported as one of the genetic risk factors of autism spectrum disorder. Thus, in this study, we used Shank2−/− (Shank2 knock-out, KO) mice to investigate the controversial pain sensitivity issue and found that Shank2 KO mice showed reduced tactile perception and analgesia to chronic pain. PMID:27145803

  10. Proteomic Analysis of Aortae from Human Lipoprotein(a) Transgenic Mice Shows an Early Metabolic Response Independent of Atherosclerosis

    PubMed Central

    Rodger, Euan J.; Suetani, Rachel J.; Jones, Gregory T.; Kleffmann, Torsten; Carne, Alan; Legge, Michael; McCormick, Sally P. A.

    2012-01-01

    Background Elevated low density lipoprotein (LDL) and lipoprotein(a) are independent risk factors for the development of atherosclerosis. Using a proteomic approach we aimed to determine early changes in arterial protein expression in transgenic mice containing both human LDL and lipoprotein(a) in circulation. Methods and Results Plasma lipid analyses showed the lipoprotein(a) transgenic mice had significantly higher lipid levels than wildtype, including a much increased LDL and high density lipoprotein (HDL) cholesterol. Analysis of aortae from lipoprotein(a) mice showed lipoprotein(a) accumulation but no lipid accumulation or foam cells, leaving the arteries essentially atherosclerosis free. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 34 arterial proteins with significantly altered abundance (P<0.05) in lipoprotein(a) transgenic mice compared to wildtype including 17 that showed a ≥2 fold difference. Some proteins of interest showed a similarly altered abundance at the transcript level. These changes collectively indicated an initial metabolic response that included a down regulation in energy, redox and lipid metabolism proteins and changes in structural proteins at a stage when atherosclerosis had not yet developed. Conclusions Our study shows that human LDL and lipoprotein(a) promote changes in the expression of a unique set of arterial proteins which may be early indicators of the metabolic disturbances preceding atherosclerosis. PMID:22276189

  11. Habituation under stress: shocked mice show nonassociative learning in a T-maze.

    PubMed

    Mitchell, D; Osborne, E W; O'Boyle, M W

    1985-03-01

    Conflicting predictions of reinforcement and neophobia-arousal theories were evaluated in a simple choice task. Four groups of C57BL/6J mice were administered daily two-trial tests in a uniform T-maze for 10 consecutive days. For three groups, the contingencies of footshock treatments were manipulated to reinforce alternation, perseveration, or both. A control group that was not administered footshock alternated, but all three groups that were stressed perseverated more and more across tests, despite the differences in reinforcement contingencies. These results are inconsistent with the predictions of reinforcement theory but consistent with the view that stressed or aroused animals are neophobic and use nonassociative learning (habituation) to distinguish between novel and familiar alternatives.

  12. MRL/MpJ mice show unique pathological features after experimental kidney injury.

    PubMed

    Shiozuru, Daichi; Ichii, Osamu; Kimura, Junpei; Nakamura, Teppei; Elewa, Yaser Hosny Ali; Otsuka-Kanazawa, Saori; Kon, Yasuhiro

    2016-02-01

    Clarification of the renal repair process is crucial for developing novel therapeutic strategies for kidney injury. MRL/MpJ mice have a unique repair process characterized by low scar formation. The pathological features of experimentally injured MRL/MpJ and C57BL/6 mouse kidneys were compared to examine the renal repair process. The dilation and atrophy of renal tubules were observed in folic acid (FA)-induced acute kidney injury (AKI) in both strains, and the histopathological injury scores and number of interleukin (IL)-1F6-positive damaged distal tubules and kidney injury molecule 1 (KIM-1)-positive damaged proximal tubules drastically increased 1 day after AKI induction. However, KIM-1-positive tubules and the elevation of serum renal function markers were significantly fewer and lower, respectively, in MRL/MpJ mice at days 2 and 7 after AKI. After traumatic kidney injury (TKI) via needle puncture, severe tubular necrotic lesions in the punctured area and fibrosis progressed in both strains. Indices for fibrosis such as aniline blue-positive area, number of alpha smooth muscle actin-positive myofibroblasts, and messenger RNA expression levels of Tgfb1 and Mmp2 indicated lower fibrotic activity in MRL/MpJ kidneys. Characteristically, only MRL/MpJ kidneys manifested remarkable calcification around the punctured area beginning 7 days after TKI. The pathological features of injured MRL/MpJ and C57BL/6 kidneys differed, especially those of kidneys with mild proximal tubular injuries after FA-induced AKI. Lower fibrotic activity and increased calcification after TKI were observed in MRL/MpJ kidneys. These findings clarified the unique pathological characteristics of MRL/MpJ mouse kidneys and contribute to understanding of the renal repair process after kidney injury.

  13. Nanoemulsified green tea extract shows improved hypocholesterolemic effects in C57BL/6 mice.

    PubMed

    Kim, Young Jun; Houng, Soung-Jin; Kim, Jae Hoon; Kim, Young-Rok; Ji, Hong Geun; Lee, Sung-Joon

    2012-02-01

    Nanoemulsification of nutrients could improve bioavailability by enhancing intestinal uptake. We investigated the antioxidant and hypolipidemic effects of nanoemulsified green tea extract (NGTE). Antioxidant effect was measured by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assay and dichlorofluorescein diacetate (DCFH-DA) assay. C57BL/6 mice were fed a control high-fat diet, green tea extract (GTE), or NGTE diet for 4 weeks. In composition analysis, GTE and NGTE contained similar total catechin concentrations. The antioxidative effect of GTE was comparable with that of NGTE. In the ABTS assay, GTE had a marked effect, although NGTE was more effective than GTE in the DCFH-DA assay. In the mouse feeding experiment, total and low-density lipoprotein (LDL) cholesterol concentrations were significantly reduced after NGTE treatment in comparison with GTE treatment in high-fat-fed C57BL/6J mice over the course of 4 weeks. The hypocholesterolemic effects were greater in the NGTE group compared with the GTE group (24% vs. 15.4% LDL cholesterol reduction compared with the control). Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase was significantly down-regulated. Protein expression of LDL receptor was significantly increased in the livers of both the GTE- and NGTE-treated groups (+234.1%, P<.01 and +274.7%, P<.001), with a greater effect in the NGTE than in the GTE group. Cholesterol 7α-hydroxylase gene expression was similarly increased in both the GTE and NGTE groups. These results suggest that nanoemulsification significantly increased hypocholesterolemic effects of GTE in vivo due to increased bioavailability.

  14. Aged APP23 mice show a delay in switching to the use of a strategy in the Barnes maze.

    PubMed

    Prut, L; Abramowski, D; Krucker, T; Levy, C L; Roberts, A J; Staufenbiel, M; Wiessner, C

    2007-04-16

    Spatial learning and memory deficits in the APP23 transgenic mice have mainly been studied using the Morris water maze (MWM). However learning in the MWM relies on swimming abilities and may be confounded by the stressful nature of this test. We have therefore assessed spatial learning and memory in 12-month-old APP23 using a dry-land maze test developed by Barnes. Mice were given daily learning trials for a total of 41 successive days. After a 12-day interval the mice were re-tested for 4 additional days in order to examine the spatial memory retention. Immediately following this phase, reversal learning was examined for 13 additional days by moving the escape tunnel to the opposite position. During the initial learning phase, APP23 mice showed a significantly longer latency to find the escape tunnel as well as an increased number of errors compared to non-transgenic littermates. These deficits appeared to be due to a delay in switching from a "no strategy" to a spatial strategy. Indeed, this same delay in the use of spatial strategy was observed in the reversal phase of the study. Our results suggest that impairments in APP23 mice in learning and memory maze tests may be due to a specific deficit in the use of spatial strategy.

  15. Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.

    PubMed

    Danglot, Lydia; Zylbersztejn, Kathleen; Petkovic, Maja; Gauberti, Maxime; Meziane, Hamid; Combe, Roy; Champy, Marie-France; Birling, Marie-Christine; Pavlovic, Guillaume; Bizot, Jean-Charles; Trovero, Fabrice; Della Ragione, Floriana; Proux-Gillardeaux, Véronique; Sorg, Tania; Vivien, Denis; D'Esposito, Maurizio; Galli, Thierry

    2012-02-08

    Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

  16. CD38 Knockout Mice Show Significant Protection Against Ischemic Brain Damage Despite High Level Poly-ADP-Ribosylation.

    PubMed

    Long, Aaron; Park, Ji H; Klimova, Nina; Fowler, Carol; Loane, David J; Kristian, Tibor

    2017-01-01

    Several enzymes in cellular bioenergetics metabolism require NAD(+) as an essential cofactor for their activity. NAD(+) depletion following ischemic insult can result in cell death and has been associated with over-activation of poly-ADP-ribose polymerase PARP1 as well as an increase in NAD(+) consuming enzyme CD38. CD38 is an NAD(+) glycohydrolase that plays an important role in inflammatory responses. To determine the contribution of CD38 activity to the mechanisms of post-ischemic brain damage we subjected CD38 knockout (CD38KO) mice and wild-type (WT) mice to transient forebrain ischemia. The CD38KO mice showed a significant amelioration in both histological and neurologic outcome following ischemic insult. Decrease of hippocampal NAD(+) levels detected during reperfusion in WT mice was only transient in CD38KO animals, suggesting that CD38 contributes to post-ischemic NAD(+) catabolism. Surprisingly, pre-ischemic poly-ADP-ribose (PAR) levels were dramatically higher in CD38KO animals compared to WT animals and exhibited reduction post-ischemia in contrast to the increased levels in WT animals. The high PAR levels in CD38 mice were due to reduced expression levels of poly-ADP-ribose glycohydrolase (PARG). Thus, the absence of CD38 activity can not only directly affect inflammatory response, but also result in unpredicted alterations in the expression levels of enzymes participating in NAD(+) metabolism. Although the CD38KO mice showed significant protection against ischemic brain injury, the changes in enzyme activity related to NAD(+) metabolism makes the determination of the role of CD38 in mechanisms of ischemic brain damage more complex.

  17. Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection▿ †

    PubMed Central

    Medina, Freddy A.; de Almeida, Cecilia J.; Dew, Elliott; Li, Jiangwei; Bonuccelli, Gloria; Williams, Terence M.; Cohen, Alex W.; Pestell, Richard G.; Frank, Philippe G.; Tanowitz, Herbert B.; Lisanti, Michael P.

    2006-01-01

    A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1−/− mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1−/− mice. However, infection of Cav-1−/− macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1−/− mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1−/− mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1−/− mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1−/− macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium. PMID:16982844

  18. PACAP-deficient mice show attenuated corticosterone secretion and fail to develop depressive behavior during chronic social defeat stress

    PubMed Central

    Lehmann, Michael L.; Mustafa, Tomris; Eiden, Adrian M.; Herkenham, Miles; Eiden, Lee E.

    2012-01-01

    Summary The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis and the adrenal gland in response to various stressors. We previously found that in response to acute psychological stress (restraint), elevated corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamic paraventricular nucleus (PVN) as well as elevated plasma corticosterone (CORT) were profoundly attenuated in PACAP-deficient mice. To determine whether HPA axis responses and stress-induced depressive-like behaviors in a chronic stress paradigm are affected by PACAP deficiency, we subjected mice to 14 days of social defeat stress. Defeat-exposed PACAP−/− mice showed a marked attenuation of stress-induced increases in serum CORT levels, cellular PVN ΔFosB immunostaining, and depressive-like behaviors (social interaction and forced swim tests) compared to wild-type control mice. The PACAP−/− mice showed reduced PVN FosB-positive cell numbers, but relatively elevated cell counts in several forebrain areas including the medial prefrontal cortex, after social stress. PACAP appears to be specific for mediating HPA activation only in psychological stress because marked elevations in plasma CORT after a systemic stressor (lipopolysaccharide administration) occurred regardless of genotype. We conclude that chronically elevated CORT is a key component of depressive effects of social defeat, and that attenuation of the CORT response at the level of the PVN, as well as extrahypothalamic forebrain regions, in PACAP-deficient mice protects from development of depressive behavior. PMID:23062748

  19. Coumarin compounds of Biebersteinia multifida roots show potential anxiolytic effects in mice

    PubMed Central

    2013-01-01

    Background Traditional preparations of the root of Biebersteinia multifida DC (Geraniaceae), a native medicinal plant of Irano-Turanian floristic region, have been used for the treatment of phobias as anxiolytic herbal preparation. Methods We utilized the phobic behavior of mice in an elevated plus-maze as a model to evaluate the anxiolytic effect of the plant extract and bio-guided fractionation was applied to isolate the active compounds. Total root extract, alkaline and ether fraction were administered to mice at different doses 30 and 90 min prior to the maze test. Saline and diazepam were administered as negative and positive controls, respectively. The time spent in open and closed arms, an index of anxiety behavior and entry time, was measured as an index of animal activity. Results The total root extract exhibited anxiolytic effect which was comparable to diazepam but with longer duration. This sustained effect of the crude extract was sustained for 90 min and was even more after injection of 45 mg/kg while the effect of diazepam had been reduced by 90 min. The anxiolytic effect factor was only present in the alkaline fraction and displayed its effect at lower doses than diazepam while pure vasicinone as the previously known alkaloid did not shown anxiolytic effect. The effect of the alkaline fraction was in a dose dependent manner starting at 0.2 mg/kg with a maximum at 1.0 mg/kg. Bio-guided fractionation using a variety of chromatographic methods led to isolation and purification of three coumarin derivatives from the bioactive fraction, including umbelliferone, scopoletin, and ferulic acid. Conclusion For the first time, bio-guided fractionation of the root extract of B. multifida indicates significant sustained anxiolytic effects which led to isolation of three coumarin derivatives with well-known potent MAO inhibitory and anti-anxiety effects. These data contribute to evidence-based traditional use of B. multifida root for anxiety disorders. PMID

  20. Histomorphometric Parameters of the Growth Plate and Trabecular Bone in Wild-Type and Trefoil Factor Family 3 (Tff3)-Deficient Mice Analyzed by Free and Open-Source Image Processing Software.

    PubMed

    Bijelić, Nikola; Belovari, Tatjana; Stolnik, Dunja; Lovrić, Ivana; Baus Lončar, Mirela

    2017-08-01

    Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.

  1. Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

    NASA Technical Reports Server (NTRS)

    Fritzsch, B.; Signore, M.; Simeone, A.

    2001-01-01

    We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

  2. Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

    NASA Technical Reports Server (NTRS)

    Fritzsch, B.; Signore, M.; Simeone, A.

    2001-01-01

    We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

  3. Adolescent C57BL/6J mice show elevated alcohol intake, but reduced taste aversion, as compared to adult mice: a potential behavioral mechanism for binge drinking

    PubMed Central

    Holstein, Sarah E.; Spanos, Marina; Hodge, Clyde W.

    2011-01-01

    Background Binge alcohol drinking during adolescence is a serious health problem which may increase future risk of an alcohol use disorder. Although there are several different procedures by which to preclinically model binge-like alcohol intake, limited-access procedures offer the advantage of achieving high voluntary alcohol intake and pharmacologically relevant blood alcohol concentrations (BACs). Therefore, in the current study, developmental differences in binge-like alcohol drinking using a limited-access cycling procedure were examined. In addition, as alcohol drinking has been negatively correlated with sensitivity to the aversive properties of alcohol, we examined developmental differences in sensitivity to an alcohol-induced conditioned taste aversion (CTA). Methods Binge-like alcohol consumption was investigated in adolescent (4 wk) and adult (10 wk) male C57BL/6J mice for 2-4 h/day for 16 d. Developmental differences in sensitivity to an alcohol-induced CTA were examined in adolescent and adult mice, with saline or alcohol (3 or 4 g/kg) repeatedly paired with intake of a novel tastant (NaCl). Results Adolescent mice showed a significant increase in alcohol intake as compared to adults, with adolescents achieving higher BACs and increasing alcohol consumption over successive cycles of the binge procedure. Conversely, adolescent mice exhibited a dose-dependent reduction in sensitivity to the aversive properties of alcohol, as compared to adult mice, with adolescent mice failing to develop a CTA to 3 g/kg alcohol. Finally, extinction of an alcohol CTA was observed following conditioning with a higher dose of alcohol in adolescent, versus adult, mice. Conclusions These results indicate that adolescent mice consume more alcohol, per kg body weight, than adults in a binge-like model of alcohol drinking, and demonstrate a blunted sensitivity to the conditioned aversive effects of alcohol. Overall, this supports a behavioral framework by which heightened binge

  4. Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss

    PubMed Central

    Seyrantepe, Volkan; Lema, Pablo; Caqueret, Aurore; Dridi, Larbi; Bel Hadj, Samar; Carpentier, Stephane; Boucher, Francine; Levade, Thierry; Carmant, Lionel; Gravel, Roy A.; Hamel, Edith; Vachon, Pascal; Di Cristo, Graziella; Michaud, Jacques L.; Morales, Carlos R.; Pshezhetsky, Alexey V.

    2010-01-01

    Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4 −/−;Hexa −/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa −/− or Neu4 −/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating GM2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa −/− mice. PMID:20862357

  5. Mice doubly-deficient in lysosomal hexosaminidase A and neuraminidase 4 show epileptic crises and rapid neuronal loss.

    PubMed

    Seyrantepe, Volkan; Lema, Pablo; Caqueret, Aurore; Dridi, Larbi; Bel Hadj, Samar; Carpentier, Stephane; Boucher, Francine; Levade, Thierry; Carmant, Lionel; Gravel, Roy A; Hamel, Edith; Vachon, Pascal; Di Cristo, Graziella; Michaud, Jacques L; Morales, Carlos R; Pshezhetsky, Alexey V

    2010-09-16

    Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-);Hexa(-/-)) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/-) or Neu4(-/-) siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2) ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/-) mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/-) mice.

  6. Transgenic Mice with Increased Astrocyte Expression of IL-6 Show Altered Effects of Acute Ethanol on Synaptic Function

    PubMed Central

    Hernandez, Ruben V.; Puro, Alana C.; Manos, Jessica C.; Huitron-Resendiz, Salvador; Reyes, Kenneth C.; Liu, Kevin; Vo, Khanh; Roberts, Amanda J.; Gruol, Donna L.

    2015-01-01

    A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including

  7. Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function.

    PubMed

    Hernandez, Ruben V; Puro, Alana C; Manos, Jessica C; Huitron-Resendiz, Salvador; Reyes, Kenneth C; Liu, Kevin; Vo, Khanh; Roberts, Amanda J; Gruol, Donna L

    2016-04-01

    A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including

  8. Atoh1 Null Mice Show Directed Afferent Fiber Growth to Undifferentiated Ear Sensory Epithelia Followed by Incomplete Fiber Retention

    PubMed Central

    Fritzsch, B.; Matei, V.A.; Nichols, D.H.; Bermingham, N.; Jones, K.; Beisel, K.W.; Wang, V.Y.

    2005-01-01

    Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain-derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1- or Bdnf-mediated β-galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1- and BDNF-β-galactosidase–positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1-β-galactosidase–positive but only occasional Bdnf-β-galactosidase–positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf-β-galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1-β-galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1- and Bdnf-β-galactosidase–positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf-β-galactosidase–positive cells. Few fibers remain to the saccule, utricle, and the base of

  9. Elevated blood pressure, heart rate and body temperature in mice lacking the XLαs protein of the Gnas locus is due to increased sympathetic tone

    PubMed Central

    Nunn, Nicolas; Feetham, Claire H; Martin, Jennifer; Barrett-Jolley, Richard; Plagge, Antonius

    2013-01-01

    New Findings What is the central question of this study? Previously, we showed that Gnasxl knock-out mice are lean and hypermetabolic, with increased sympathetic stimulation of adipose tissue. Do these mice also display elevated sympathetic cardiovascular tone? Is the brain glucagon-like peptide-1 system involved? What is the main finding and its importance? Gnasxl knock-outs have increased blood pressure, heart rate and body temperature. Heart rate variability analysis suggests an elevated sympathetic tone. The sympatholytic reserpine had stronger effects on blood pressure, heart rate and heart rate variability in knock-out compared with wild-type mice. Stimulation of the glucagon-like peptide-1 system inhibited parasympathetic tone to a similar extent in both genotypes, with a stronger associated increase in heart rate in knock-outs. Deficiency of Gnasxl increases sympathetic cardiovascular tone. Imbalances of energy homeostasis are often associated with cardiovascular complications. Previous work has shown that Gnasxl-deficient mice have a lean and hypermetabolic phenotype, with increased sympathetic stimulation of adipose tissue. The Gnasxl transcript from the imprinted Gnas locus encodes the trimeric G-protein subunit XLαs, which is expressed in brain regions that regulate energy homeostasis and sympathetic nervous system (SNS) activity. To determine whether Gnasxl knock-out (KO) mice display additional SNS-related phenotypes, we have now investigated the cardiovascular system. The Gnasxl KO mice were ∼20 mmHg hypertensive in comparison to wild-type (WT) littermates (P≤ 0.05) and hypersensitive to the sympatholytic drug reserpine. Using telemetry, we detected an increased waking heart rate in conscious KOs (630 ± 10 versus 584 ± 12 beats min−1, KO versus WT, P≤ 0.05). Body temperature was also elevated (38.1 ± 0.3 versus 36.9 ± 0.4°C, KO versus WT, P≤ 0.05). To investigate autonomic nervous system influences, we used heart rate variability

  10. The delta 6 desaturase knock out mouse reveals that immunomodulatory effects of essential n-6 and n-3 polyunsaturated fatty acids are both independent of and dependent upon conversion.

    PubMed

    Monk, Jennifer M; Liddle, Danyelle M; Cohen, Daniel J A; Tsang, Denis H; Hillyer, Lyn M; Abdelmagid, Salma A; Nakamura, Manabu T; Power, Krista A; Ma, David W L; Robinson, Lindsay E

    2016-06-01

    Typically fatty acids (FA) exert differential immunomodulatory effects with n-3 [α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and n-6 [linoleic acid (LA) and arachidonic acid (AA)] exerting anti- and pro-inflammatory effects, respectively. This over-simplified interpretation is confounded by a failure to account for conversion of the parent FA (LA and ALA) to longer-chain bioactive products (AA and EPA/DHA, respectively), thereby precluding discernment of the immunomodulatory potential of specific FA. Therefore, we utilized the Δ6-desaturase model, wherein knockout mice (D6KO) lack the Fads2 gene encoding for the rate-limiting enzyme that initiates FA metabolism, thereby providing a model to determine specific FA immunomodulatory effects. Wild-type (WT) and D6KO mice were fed one of four isocaloric diets differing in FA source (9weeks): corn oil (LA-enriched), arachidonic acid single cell oil (AA-enriched), flaxseed oil (ALA-enriched) or menhaden fish oil (EPA/DHA-enriched). Splenic mononuclear cell cytokine production in response to lipopolysaccharide (LPS), T-cell receptor (TCR) and anti-CD40 stimulation was determined. Following LPS stimulation, AA was more bioactive compared to LA, by increasing inflammatory cytokine production of IL-6 (1.2-fold) and TNFα (1.3-fold). Further, LPS-stimulated IFNγ production in LA-fed D6KO mice was reduced 5-fold compared to LA-fed WT mice, indicating that conversion of LA to AA was necessary for cytokine production. Conversely, ALA exerted an independent immunomodulatory effect from EPA/DHA and all n-3 FA increased LPS-stimulated IL-10 production versus LA and AA. These data definitively identify specific immunomodulatory effects of individual FA and challenge the simplified view of the immunomodulatory effects of n-3 and n-6 FA.

  11. Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

    PubMed

    Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

    2017-05-01

    Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  12. Ilexgenin A, a novel pentacyclic triterpenoid extracted from Aquifoliaceae shows reduction of LPS-induced peritonitis in mice.

    PubMed

    Sun, Weidong; Liu, Chang; Zhang, Yaqi; Qiu, Xia; Zhang, Li; Zhao, Hongxia; Rong, Yi; Sun, Yun

    2017-02-15

    Ilexgenin A (IA) is a novel pentacyclic triterpenoid, which extracted from leaves of Ilex hainanensis Merr. In the present study, we aim to explore anti-inflammatory activity of IA on LPS-induced peritonitis and its underlying molecular mechanism. The results determined that IA was capable of suppressing peritonitis in mice induced by intraperitoneal (i.p.) injection of lipopolysaccaride (LPS). Furthermore, the results showed that IA dramatically inhibited levels of inflammatory cells infiltration in peritoneal cavity and serum in LPS-induced mice peritonitis model. Besides, IA could dramatically inhibit levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in peritoneal cavity in LPS-induced mice peritonitis model. In vitro study, the results showed that IA inhibited production of IL-1β, IL-6 and TNF-α at transcriptional and translational levels in RAW 264.7 cells induced by LPS. Furthermore, IA could suppress the LPS-induced activation of Akt and downstream degradation and phosphorylation of kappa B-α (IκB-α). Moreover, IA could significantly inhibit ERK 1/2 phosphorylation in RAW 264.7 cells induced by LPS. These results were concurrent with molecular docking which revealed ERK1/2 inhibition. These results demonstrated that IA might as an anti-inflammatory agent candidate for inflammatory disease therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Mice devoid of interferon regulatory factor 1 (IRF-1) show normal expression of type I interferon genes.

    PubMed Central

    Reis, L F; Ruffner, H; Stark, G; Aguet, M; Weissmann, C

    1994-01-01

    The transcription factor interferon regulatory factor 1 (IRF-1) binds tightly to the interferon (IFN)-beta promoter and has been implicated in the induction of type I IFNs. We generated mice devoid of functional IRF-1 by targeted gene disruption. As reported by others, IRF-1-deficient mice showed a discrete phenotype: the CD4/CD8 ratio was increased and IFN-gamma-induced levels of macrophage iNO synthase mRNA were strongly diminished. However, type I IFN induction in vivo by virus or double-stranded RNA was unimpaired, as evidenced by serum IFN titers and IFN mRNA levels in spleen, liver and lung. There was also no impairment in the response of type I IFN-inducible genes. Therefore, IRF-1 is not essential for these processes in vivo. Images PMID:7957048

  14. Chronic Methadone Treatment Shows a Better Cost/Benefit Ratio than Chronic Morphine in Mice

    PubMed Central

    Enquist, Johan; Ferwerda, Madeline; Milan-Lobo, Laura

    2012-01-01

    Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading μ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not. PMID:22062352

  15. Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

    PubMed

    Enquist, Johan; Ferwerda, Madeline; Milan-Lobo, Laura; Whistler, Jennifer L

    2012-02-01

    Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading μ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

  16. Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance.

    PubMed

    Talukder, M A Hassan; Preda, Marilena; Ryzhova, Larisa; Prudovsky, Igor; Pinz, Ilka M

    2016-03-01

    Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin-3 (CAV3). We further test whether haploinsufficiency forCAV3 increases the susceptibility to high-fat-induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of theIR We show that haploinsufficiency forCAV3 increases susceptibility to palmitate-induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show thatCAV3 andIRdirectly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin-stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin-stimulated glucose uptake inHL-1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin-independent serine phosphorylation ofIRS-1 by 35%. In addition, we found lipid induced downregulation ofCD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss ofCAV3 interferes with downstream insulin signaling and lipid uptake, implicatingCAV3 as a regulator of theIRand regulator of lipid uptake in the heart. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  17. Plgf-/-eNos-/- mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia.

    PubMed

    Gigante, Bruna; Morlino, Giulia; Gentile, Maria Teresa; Persico, Maria Graziella; De Falco, Sandro

    2006-05-01

    Neo-angiogenesis is a complex phenomenon modulated by the concerted action of several molecular factors. We have generated a congenic line of knockout mice carrying null mutations of both placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS), two genes that play a pivotal role in the regulation of pathological angiogenesis. In the present study, we describe the phenotype of this new experimental animal model after surgically induced hind-limb ischemia. Plgf-/-, eNos-/-, Plgf-/- eNos-/-, and wild-type C57BL/6J mice were studied. Plgf-/- eNos-/- mice showed the most severe phenotype: self-amputation, and death occurred in up to 47% of the animals studied; in ischemic legs, capillary density was severely reduced; macrophage infiltration and oxidative stress increased as compared to the other groups of animals. These changes were associated with an up-regulation of both inducible NOS (iNOS) expression and vascular endothelial growth factor (VEGF) protein levels in ischemic limbs, and to an increased extent of protein nitration. Our results demonstrate that the deletion of these two genes, Plgf, which acts in synergism with VEGF, and eNos, a downstream mediator of VEGF, determines a significant change in the vascular response to an ischemic stimulus and that oxidative stress within the ischemic tissue represents a crucial factor to maintain tissue homeostasis.

  18. Transgenic mice with ectopic expression of constitutively active TLR4 in adipose tissues do not show impaired insulin sensitivity.

    PubMed

    Ono-Moore, Kikumi D; Zhao, Ling; Huang, Shurong; Kim, Jeonga; Rutkowsky, Jennifer M; Snodgrass, Ryan G; Schneider, Dina A; Quon, Michael J; Graham, James L; Havel, Peter J; Hwang, Daniel H

    2017-08-04

    Chronic low-grade inflammation is associated with obesity and diabetes. However, what causes and mediates chronic inflammation in metabolic disorders is not well understood. Toll-like receptor 4 (TLR4) mediates both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns and endogenous molecules, respectively. Saturated fatty acids can activate TLR4, and TLR4-deficient mice were protected from high fat diet (HFD)-induced obesity and insulin resistance, suggesting that TLR4-mediated inflammation may cause metabolic dysfunction, such as obesity and insulin resistance. We generated two transgenic (TG) mouse lines expressing a constitutively active TLR4 in adipose tissue and determined whether these TG mice would show increased insulin resistance. TG mice fed a high fat or a normal chow diet did not exhibit increased insulin resistance compared to their wild-type controls despite increased localized inflammation in white adipose tissue. Furthermore, females of one TG line fed a normal chow diet had improved insulin sensitivity with reduction in both adiposity and body weight when compared with wild-type littermates. There were significant differences between female and male mice in metabolic biomarkers and mRNA expression in proinflammatory genes and negative regulators of TLR4 signaling, regardless of genotype and diet. Together, these results suggest that constitutively active TLR4-induced inflammation in white adipose tissue is not sufficient to induce systemic insulin resistance, and that high fat diet-induced insulin resistance may require other signals in addition to TLR4-mediated inflammation. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

  19. The APOE4 allele shows opposite sex bias in microbleeds and Alzheimer’s Disease of humans and mice

    PubMed Central

    Cacciottolo, Mafalda; Christensen, Amy; Moser, Alexandra; Liu, Jiahui; Pike, Christian J.; Sullivan, Patrick M.; Morgan, Todd E.; Dolzhenko, Egor; Charidimou, Andreas; Wahlund, Lars-Olaf; Wiberg, Maria Kristofferson; Shams, Sara; Chiang, Gloria Chia-Yi; Finch, Caleb E.

    2015-01-01

    The APOE4 allele confers greater risk of Alzheimer’s Disease (AD) for women than men, in conjunction with greater clinical deficits per unit of AD neuropathology (plaques, tangles). Cerebral microbleeds, which contribute to cognitive dysfunctions during AD, also show APOE4 excess, but sex-APOE allele interactions are not described. We report that elderly men diagnosed for mild cognitive impairment (MCI) and AD showed a higher risk of cerebral cortex microbleeds with APOE4 allele dose effect in two clinical cohorts (ADNI and KIDS). Sex-APOE interactions were further analyzed in EFAD mice carrying human APOE alleles and familial AD genes. At 7 months, E4FAD mice had cerebral cortex microbleeds with female excess, in contrast to humans. Cerebral amyloid angiopathy (CAA), plaques, and soluble Aβ also showed female excess. Both the cerebral microbleeds and CAA increased in proportion to individual Aβ load. In humans, the opposite sex bias of APOE4 allele for microbleeds vs the plaques and tangles is the first example of organ-specific, sex-linked APOE allele effects, and further shows AD as a uniquely human condition. PMID:26686669

  20. A novel BET bromodomain inhibitor, RVX-208, shows reduction of atherosclerosis in hyperlipidemic ApoE deficient mice.

    PubMed

    Jahagirdar, Ravi; Zhang, Haiyan; Azhar, Salman; Tobin, Jennifer; Attwell, Sarah; Yu, Raymond; Wu, Jin; McLure, Kevin G; Hansen, Henrik C; Wagner, Gregory S; Young, Peter R; Srivastava, Rai Ajit K; Wong, Norman C W; Johansson, Jan

    2014-09-01

    Despite the benefit of statins in reducing cardiovascular risk, a sizable proportion of patients still remain at risk. Since HDL reduces CVD risk through a process that involves formation of pre-beta particles that facilitates the removal of cholesterol from the lipid-laden macrophages in the arteries, inducing pre-beta particles, may reduce the risk of CVD. A novel BET bromodomain antagonist, RVX-208, was reported to raise apoA-I and increase preβ-HDL particles in non-human primates and humans. In the present study, we investigated the effect of RVX-208 on aortic lesion formation in hyperlipidemic apoE(-/-) mice. Oral treatments of apoE(-/-) mice with 150 mg/kg b.i.d RVX-208 for 12 weeks significantly reduced aortic lesion formation, accompanied by 2-fold increases in the levels of circulating HDL-C, and ∼50% decreases in LDL-C, although no significant changes in plasma apoA-I were observed. Circulating adhesion molecules as well as cytokines also showed significant reduction. Haptoglobin, a proinflammatory protein, known to bind with HDL/apoA-I, decreased >2.5-fold in the RVX-208 treated group. With a therapeutic dosing regimen in which mice were fed Western diet for 10 weeks to develop lesions followed by switching to a low fat diet and concurrent treatment with RVX-208 for 14 weeks, RVX-208 similarly reduced lesion formation by 39% in the whole aorta without significant changes in the plasma lipid parameters. RVX-208 significantly reduced the proinflammatory cytokines IP-10, MIP1(®) and MDC. These results show that the antiatherogenic activity of BET inhibitor, RVX-208, occurs via a combination of lipid changes and anti-inflammatory activities. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Dynamic studies of positron-emitting putative tumor marker /sup 132/Cs in mice show differential tumor and regional uptake

    SciTech Connect

    McKee, J.S.; Durocher, J.J.; Bose, R.; Gusdal, M.I.; Sharma, G.P.; Pinsky, C.; Gallop, D.

    1985-01-01

    Positron-emitting /sup 132/Cs (t1/2 = 6.47 days) was generated from stable /sup 133/CsCl via the /sup 133/Cs (p,pn) /sup 132/Cs reaction. BALB/c mice, bearing implanted MT296 mammary tumors, were given 4.6 mEq kg-1 of /sup 132/CsCl via a single intraperitoneal injection. Postinjection uptake of /sup 132/Cs into body regions was monitored in vivo with external detectors. Positron emission from the tumor region was continuously greater than that from the head, the numerical ratio of mean emission intensities being fourfold at 10 min postinjection. Tissues excised from these mice postmortem showed sequence of relative tissue cesium uptake rates to be kidney 1.8, small intestine 1.7, tumor 1.0, skin 0.75, liver 0.75, skeletal muscle 0.4, and brain 0.28. Comparative studies with multiple injections of stable cesium and rubidium showed this sequence to be ion-specific. These observations suggest that positron-emitting isotopes of cesium could provide useful markers for tumors of several tissues.

  2. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice.

    PubMed

    Hanna, Zaher; Priceputu, Elena; Hu, Chunyan; Vincent, Patrick; Jolicoeur, Paul

    2006-03-01

    HIV-1 Nef has the ability to downmodulate CD4 cell surface expression. Several studies have shown that CD4 downregulation is required for efficient virus replication and high infectivity. However, the pathophysiological relevance of this phenomenon in vivo, independently of its role in sustaining high virus loads, remains unclear. We studied the impact of the CD4 downregulation function of Nef on its pathogenesis in vivo, in the absence of viral replication, in the CD4C/HIV transgenic (Tg) mouse model. Two independent Nef mutants (RD35/36AA and D174K), known to abrogate CD4 downregulation, were tested in Tg mice. Flow cytometry analysis showed that downregulation of murine CD4 was severely decreased or abrogated on Tg T cells expressing respectively Nef(RD35/36AA) and Nef(D174K). Similarly, the severe depletion of double-positive CD4+CD8+ and of single-positive CD4+CD8- thymocytes, usually observed with Nef(Wt), was not detected in Nef(RD35/36AA) and Nef(D174K) Tg mice. However, both mutant Tg mice showed a partial depletion of peripheral CD4+ T cells. This was accompanied, as previously reported for Net(Wt) Tg mice, by the presence of an activated/memory-like phenotype (CD69+, CD25+, CD44+, CD45RB(Low), CD62(Low)) of CD4+ T cells expressing Nef(RD35/36AA) and to a lesser extent Nef(D174K). In addition, both mutants retained the ability to block CD4+ T cell proliferation in vitro after anti-CD3 stimulation, but not to enhance apoptosis/death of CD4+ T cells. Therefore, it appears that Nef-mediated CD4 downregulation is associated with thymic defects, but segregates independently of the activated/memory-like phenotype, of the partial depletion and of the impaired in vitro proliferation of peripheral CD4+ T cells. Histopathological assessment revealed the total absence of or decrease severity and frequency of organ AIDS-like diseases (lung, heart and kidney pathologies) in respectively Nef(RD35/36AA) and Nef(D174K) Tg mice, relative to those developing in Nef(Wt) Tg

  3. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice

    SciTech Connect

    Hanna, Zaher . E-mail: Zaher.Hanna@ircm.qc.ca; Priceputu, Elena; Hu, Chunyan; Vincent, Patrick; Jolicoeur, Paul

    2006-03-01

    HIV-1 Nef has the ability to downmodulate CD4 cell surface expression. Several studies have shown that CD4 downregulation is required for efficient virus replication and high infectivity. However, the pathophysiological relevance of this phenomenon in vivo, independently of its role in sustaining high virus loads, remains unclear. We studied the impact of the CD4 downregulation function of Nef on its pathogenesis in vivo, in the absence of viral replication, in the CD4C/HIV transgenic (Tg) mouse model. Two independent Nef mutants (RD35/36AA and D174K), known to abrogate CD4 downregulation, were tested in Tg mice. Flow cytometry analysis showed that downregulation of murine CD4 was severely decreased or abrogated on Tg T cells expressing respectively Nef{sup RD35/36AA} and Nef{sup D174K}. Similarly, the severe depletion of double-positive CD4{sup +}CD8{sup +} and of single-positive CD4{sup +}CD8{sup -} thymocytes, usually observed with Nef{sup Wt}, was not detected in Nef{sup RD35/36AA} and Nef{sup D174K} Tg mice. However, both mutant Tg mice showed a partial depletion of peripheral CD4{sup +} T cells. This was accompanied, as previously reported for Net{sup Wt} Tg mice, by the presence of an activated/memory-like phenotype (CD69{sup +}, CD25{sup +}, CD44{sup +}, CD45RB{sup Low}, CD62{sup Low}) of CD4{sup +} T cells expressing Nef{sup RD35/36AA} and to a lesser extent Nef{sup D174K}. In addition, both mutants retained the ability to block CD4{sup +} T cell proliferation in vitro after anti-CD3 stimulation, but not to enhance apoptosis/death of CD4{sup +} T cells. Therefore, it appears that Nef-mediated CD4 downregulation is associated with thymic defects, but segregates independently of the activated/memory-like phenotype, of the partial depletion and of the impaired in vitro proliferation of peripheral CD4{sup +} T cells. Histopathological assessment revealed the total absence of or decrease severity and frequency of organ AIDS-like diseases (lung, heart and kidney

  4. Age-Related Impairment in the 250-Millisecond Delay Eyeblink Classical Conditioning Procedure in C57BL/6 Mice

    PubMed Central

    Vogel, Richard W.; Ewers, Michael; Ross, Charlene; Gould, Thomas J.; Woodruff-Pak, Diana S.

    2002-01-01

    In this study we tested 4-, 9-, 12-, and 18-month-old C57BL/6 mice in the 250-msec delay eyeblink classical conditioning procedure to study age-related changes in a form of associative learning. The short life expectancy of mice, complete knowledge about the mouse genome, and the availability of transgenic and knock-out mouse models of age-related impairments make the mouse an excellent species for expanding knowledge on the neurobiologically and behaviorally well-characterized eyeblink classical conditioning paradigm. Based on previous research with delay eyeblink conditioning in rabbits and humans, we predicted that mice would be impaired on this cerebellar-dependent associative learning task in middle-age, at ∼9 months. To fully examine age differences in behavior in mice, we used a battery of additional behavioral measures with which to compare young and older mice. These behaviors included the acoustic startle response, prepulse inhibition, rotorod, and the Morris water maze. Mice began to show impairment in cerebellar-dependent tasks such as rotorod and eyeblink conditioning at 9 to 12 months of age. Performance in hippocampally dependent tasks was not impaired in any group, including 18-month-old mice. These results in mice support results in other species, indicating that cerebellar-dependent tasks show age-related deficits earlier in adulthood than do hippocampally dependent tasks. PMID:12359840

  5. Mice lacking thyroid hormone receptor Beta show enhanced apoptosis and delayed liver commitment for proliferation after partial hepatectomy.

    PubMed

    López-Fontal, Raquel; Zeini, Miriam; Través, Paqui G; Gómez-Ferrería, Mariana; Aranda, Ana; Sáez, Guillermo T; Cerdá, Concha; Martín-Sanz, Paloma; Hortelano, Sonsoles; Boscá, Lisardo

    2010-01-14

    The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs. Mice lacking TRalpha1/TRbeta or TRbeta alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting approximately 30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRalpha1/TRbeta or TRbeta. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR. We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRbeta- or TRalpha1/TRbeta-deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRbeta in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRbeta that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating

  6. Mice Lacking Thyroid Hormone Receptor β Show Enhanced Apoptosis and Delayed Liver Commitment for Proliferation after Partial Hepatectomy

    PubMed Central

    López-Fontal, Raquel; Zeini, Miriam; Través, Paqui G.; Gómez-Ferrería, Mariana; Aranda, Ana; Sáez, Guillermo T.; Cerdá, Concha; Martín-Sanz, Paloma; Hortelano, Sonsoles; Boscá, Lisardo

    2010-01-01

    Background The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs. Methodology/Principal Findings Mice lacking TRα1/TRβ or TRβ alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting ∼30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRα1/TRβ or TRβ. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR. Conclusions/Significance We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRβ– or TRα1/TRβ–deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRβ in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRβ that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRβ contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the

  7. Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

    PubMed Central

    Schöbel, Nicole; Radtke, Debbie; Lübbert, Matthias; Gisselmann, Günter; Lehmann, Ramona; Cichy, Annika; Schreiner, Benjamin S. P.; Altmüller, Janine; Spector, Alan C.; Spehr, Jennifer; Hatt, Hanns; Wetzel, Christian H.

    2012-01-01

    Intracellular Cl− concentrations ([Cl−]i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl− is accumulated by the Na+-K+-2Cl− cotransporter 1 (NKCC1), resulting in a [Cl−]i above electrochemical equilibrium and a depolarizing Cl− efflux upon Cl− channel opening. Here, we investigate the [Cl−]i and function of Cl− in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl−]i of WT TG neurons indicated active NKCC1-dependent Cl− accumulation. Gamma-aminobutyric acid (GABA)A receptor activation induced a reduction of [Cl−]i as well as Ca2+ transients in a corresponding fraction of TG neurons. Ca2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca2+ channels (VGCCs). Ca2+ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1−/− TG neurons, but elevated under conditions of a lowered [Cl−]o suggesting a Cl−-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca2+-activated Cl− channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca2+ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca2+-activated Cl−-dependent signal amplification mechanism in TG neurons that requires intracellular Cl− accumulation by NKCC1 and the activation of CaCCs. PMID:23144843

  8. Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice

    PubMed Central

    Vernochet, Cécile; Mariot, Virginie; Gache, Vincent; Charrin, Stéphanie; Tiret, Laurent; Dumonceaux, Julie; Dupressoir, Anne; Heidmann, Thierry

    2016-01-01

    Syncytins are envelope genes from endogenous retroviruses, “captured” for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20–40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals. PMID:27589388

  9. MUCI Facilitation of Growth in Chemically Induced Mammary Gland Tumors in Muc-1 Mutant and MUCI Transgenic Mice.

    DTIC Science & Technology

    1998-08-01

    Mice PRINCIPAL INVESTIGATOR: Russell J. Vanderboom, Ph.D. CONTRACTING ORGANIZATION: Mayo Foundation Rochester, Minnesota 55905 REPORT DATE: August 1998...mucin Mucl on tumor growth in the mammary glands of mice, a chemical carcinogenic combination of medroxyprogesteone acetate (MPA) and nitroso...methylurea was administered to wildtype C57BL/6 mice and mutant knock-out C57BL/6 mice unable to express Mucl . One week after implantation of time release MPA

  10. Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

    PubMed Central

    Gey, Manuel; Wanner, Renate; Schilling, Corinna; Pedro, Maria T.; Sinske, Daniela

    2016-01-01

    Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce ‘effector’ RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types. PMID:27581653

  11. Development of a murine model for aerosolized ebolavirus infection using a panel of recombinant inbred mice.

    PubMed

    Zumbrun, Elizabeth E; Abdeltawab, Nourtan F; Bloomfield, Holly A; Chance, Taylor B; Nichols, Donald K; Harrison, Paige E; Kotb, Malak; Nalca, Aysegul

    2012-12-03

    Countering aerosolized filovirus infection is a major priority of biodefense research. Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported. A murine model of aerosolized filovirus infection in mice should be useful for screening vaccine candidates and therapies. In this study, various strains of wild-type and immunocompromised mice were exposed to aerosolized wild-type (WT) or mouse-adapted (MA) Ebola virus (EBOV). Upon exposure to aerosolized WT-EBOV, BALB/c, C57BL/6 (B6), and DBA/2 (D2) mice were unaffected, but 100% of severe combined immunodeficiency (SCID) and 90% of signal transducers and activators of transcription (Stat1) knock-out (KO) mice became moribund between 7-9 days post-exposure (dpe). Exposure to MA-EBOV caused 15% body weight loss in BALB/c, but all mice recovered. In contrast, 10-30% lethality was observed in B6 and D2 mice exposed to aerosolized MA-EBOV, and 100% of SCID, Stat1KO, interferon (IFN)-γ KO and Perforin KO mice became moribund between 7-14 dpe. In order to identify wild-type, inbred, mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 60 BXD (C57BL/6 crossed with DBA2) recombinant inbred (RI) and advanced RI (ARI) mouse strains were exposed to aerosolized MA-EBOV, and monitored for disease severity. A complete spectrum of disease severity was observed. All BXD strains lost weight but many recovered. However, infection was uniformly lethal within 7 to 12 days post-exposure in five BXD strains. Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 90-100% lethality in two strains. Analysis of post-mortem tissue from BXD strains that became moribund and were euthanized at the lower dose of MA-EBOV, showed liver damage in all mice as well as lung lesions in two of the three strains. The two

  12. Mice overexpressing 70-kDa heat shock protein show increased resistance to malonate and 3-nitropropionic acid.

    PubMed

    Dedeoglu, Alpaslan; Ferrante, Robert J; Andreassen, Ole A; Dillmann, Wolfgang H; Beal, M Flint

    2002-07-01

    Heat shock proteins (HSPs) are induced in response to oxidative stress, hypoxia-ischemia, and neuronal injury and play a protective role. Malonate and 3-nitropropionic acid (3-NP) are well-characterized animal models of Huntington's Disease (HD). They inhibit succinate dehydrogenase, inducing mitochondrial dysfunction, which triggers the generation of superoxide radicals, secondary excitotoxicity, and apoptosis. In this study, we examined whether the 70-kDa heat shock protein (HSP-70) is protective against neurotoxicity induced by malonate and 3-NP. Homozygous and heterozygous HSP-70 overexpressing mice (HSP-70+/+, HSP-70+/-) and wild-type controls received 3-NP or malonate and striatal lesion sizes were evaluated by stereology. Compared to HSP-70+/+ and HSP-70+/-, wild-type controls showed significantly larger striatal lesions following 3-NP or malonate injections. These findings support the idea that HSP-70 has a neuroprotective role that may be useful in the treatment of neurodegenerative diseases.

  13. Deep phosphorus fertiliser placement and reduced irrigation methods for rice (Oryza sativa L.) combine to knock-out competition from its nemesis, barnyard grass (Echinochloa crus-galli (L.) P.Beauv)

    USDA-ARS?s Scientific Manuscript database

    Productivity of rice is increasingly being constrained by limitations in the quantity, quality, and cost of water and nutrients, and competition from weeds. This is a ‘commentary’ on the recent work of Weerarathne et al. (2015). They reported new discoveries from greenhouse experiments that showed...

  14. Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast.

    PubMed

    Guaragnella, Nicoletta; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-08-20

    To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H(2)O(2)) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H(2)O(2) and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.

  15. SCOPRISM: a new algorithm for automatic sleep scoring in mice.

    PubMed

    Bastianini, Stefano; Berteotti, Chiara; Gabrielli, Alessandro; Del Vecchio, Flavia; Amici, Roberto; Alexandre, Chloe; Scammell, Thomas E; Gazea, Mary; Kimura, Mayumi; Lo Martire, Viviana; Silvani, Alessandro; Zoccoli, Giovanna

    2014-09-30

    Scoring of wake-sleep states by trained investigators is a time-consuming task in many sleep experiments. We aimed to validate SCOPRISM, a new open-source algorithm for sleep scoring based on automatic graphical clustering of epoch distribution. We recorded sleep and blood pressure signals of 36 orexin-deficient, 7 leptin knock-out, and 43 wild-type control mice in the PRISM laboratory. Additional groups of mice (n=14) and rats (n=6) recorded in independent labs were used to validate the algorithm across laboratories. The overall accuracy, specificity and sensitivity values of SCOPRISM (97%, 95%, and 94%, respectively) on PRISM lab data were similar to those calculated between human scorers (98%, 98%, and 94%, respectively). Using SCOPRISM, we replicated the main sleep and sleep-dependent cardiovascular findings of our previous studies. Finally, the cross-laboratory analyses showed that the SCOPRISM algorithm performed well on mouse and rat data. SCOPRISM performed similarly or even better than recently reported algorithms. SCOPRISM is a very simple algorithm, extensively (cross)validated and with the possibility to evaluate its efficacy following a quick and easy visual flow chart. We validated SCOPRISM, a new, automated and open-source algorithm for sleep scoring on a large population of mice, including different mutant strains and on subgroups of mice and rats recorded by independent labs. This algorithm should help accelerate basic research on sleep and integrative physiology in rodents. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Recombinant influenza H7 hemagglutinin containing CFLLC minidomain in the transmembrane domain showed enhanced cross-protection in mice.

    PubMed

    Wang, Yang; Zhang, Yun; Wu, Jialing; Lin, Ying; Wu, Zhihui; Wei, Ying; Wei, Xiaona; Qin, Jianru; Xue, Chunyi; Liu, George Dacai; Cao, Yongchang

    2017-09-12

    Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has been a potential pandemic threat. The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses; thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed. Our previous studies demonstrated that the HA-based structural design approach by introducing a CFLLC minidomain into transmembrane domain (TM) of H1, H5 or H9 hemagglutinin (HA) proteins by replacing with H3 subtype HA TM could enhance their cross-protection. In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM containing CFLLC minidomian by replacing its TM domain with H3 HA TM. We investigated whether introduction of CFLLC minidomain into H7 HA (H7-53TM) could increase its cross-reactivity and cross-protection against different genotypes of H7N9 viruses. The results showed that the H7-53TM either with or without squalene adjuvant induced increased HI antibodies, serum IgG antibodies, and IFN-γ production to a panel of 7 H7N9 viruses in mice. Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection (80%). Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and less pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT. In summary, this study presents a better subunit vaccine candidate (H7-53TM) against potential H7N9 pandemic. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver.

    PubMed

    Nagashima, Shuichi; Yagyu, Hiroaki; Tozawa, Ryuichi; Tazoe, Fumiko; Takahashi, Manabu; Kitamine, Tetsuya; Yamamuro, Daisuke; Sakai, Kent; Sekiya, Motohiro; Okazaki, Hiroaki; Osuga, Jun-ichi; Honda, Akira; Ishibashi, Shun

    2015-05-01

    Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.

  18. Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice.

    PubMed

    Moneta, D; Richichi, C; Aliprandi, M; Dournaud, P; Dutar, P; Billard, J M; Carlo, A S; Viollet, C; Hannon, J P; Fehlmann, D; Nunn, C; Hoyer, D; Epelbaum, J; Vezzani, A

    2002-09-01

    We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2

  19. Tolerogenic dendritic cells show gene expression profiles that are different from those of immunogenic dendritic cells in DBA/1 mice.

    PubMed

    Lee, Eun Gae; Jung, Nam-Chul; Lee, Jun-Ho; Song, Jie-Young; Ryu, Sang-Young; Seo, Han Geuk; Han, Sung Gu; Ahn, Keun Jae; Hong, Kwan Soo; Choi, Jinjung; Lim, Dae-Seog

    2016-01-01

    Tolerogenic dendritic cells (tDCs) play an important role in inducing peripheral tolerance; however, few tDC-specific markers have been identified. The aims of this study were to examine whether tDCs show a different gene expression profile from that of immunogenic DCs and identify specific gene markers of each cell type, in DBA/1 mice. tDCs were generated by treating immature DCs (imDCs) with TNF-α and type II collagen. The gene expression profiles of mature (m)DCs and tDCs were then investigated by microarray analysis and candidate markers were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Supervised selection identified 75 gene signatures, 63 of which were consistently upregulated in mDCs and 12 of which were upregulated only in tDCs. Additionally, 10 genes were overexpressed or equally expressed in both tDCs and mDCs. Scin (tDC-specific genes) and Orm1, Pdlim4 and Enpp2 (mDC-specific genes) were validated by real-time qRT-PCR. Taken together, these results clearly show that tDCs and mDCs can be identified according to their expression of specific gene markers.

  20. Severe SMA mice show organ impairment that cannot be rescued by therapy with the HDACi JNJ-26481585.

    PubMed

    Schreml, Julia; Riessland, Markus; Paterno, Mario; Garbes, Lutz; Roßbach, Kristina; Ackermann, Bastian; Krämer, Jan; Somers, Eilidh; Parson, Simon H; Heller, Raoul; Berkessel, Albrecht; Sterner-Kock, Anja; Wirth, Brunhilde

    2013-06-01

    Spinal muscular atrophy (SMA) is the leading genetic cause of early childhood death worldwide and no therapy is available today. Many drugs, especially histone deacetylase inhibitors (HDACi), increase SMN levels. As all HDACi tested so far only mildly ameliorate the SMA phenotype or are unsuitable for use in humans, there is still need to identify more potent drugs. Here, we assessed the therapeutic power of the pan-HDACi JNJ-26481585 for SMA, which is currently used in various clinical cancer trials. When administered for 64 h at 100 nM, JNJ-26481585 upregulated SMN levels in SMA fibroblast cell lines, including those from non-responders to valproic acid. Oral treatment of Taiwanese SMA mice and control littermates starting at P0 showed no overt extension of lifespan, despite mild improvements in motor abilities and weight progression. Many treated and untreated animals showed a very rapid decline or unexpected sudden death. We performed exploratory autopsy and histological assessment at different disease stages and found consistent abnormalities in the intestine, heart and lung and skeletal muscle vasculature of SMA animals, which were not prevented by JNJ-26481585 treatment. Interestingly, some of these features may be only indirectly caused by α-motoneuron function loss but may be major life-limiting factors in the course of disease. A better understanding of - primary or secondary - non-neuromuscular organ involvement in SMA patients may improve standard of care and may lead to reassessment of how to investigate SMA patients clinically.

  1. Abnormal Mammary Adipose Tissue Environment of Brca1 Mutant Mice Show a Persistent Deposition of Highly Vascularized Multilocular Adipocytes.

    PubMed

    Jones, Laundette P; Buelto, Destiney; Tago, Elaine; Owusu-Boaitey, Kwadwo E

    2011-12-08

    A major challenge to breast cancer research is the identification of alterations in the architecture and composition of the breast that are associated with breast cancer progression. The aim of the present investigation was to characterize the mammary adipose phenotype from Brca1 mutant mice in the expectation that this would shed light on the role of the mammary tissue environment in the early stages of breast tumorigenesis. We observed that histological sections of mammary tissue from adult Brca1 mutant mice abnormally display small, multilocular adipocytes that are reminiscent of brown adipose tissue (BAT) as compared to wildtype mice. Using a marker for BAT, the uncoupling protein 1 (UCP1), we demonstrated that these multilocular adipose regions in Brca1 mutant mice stain positive for UCP1. Transcriptionally, UCP1 mRNA levels in the Brca1 mutant mice were elevated greater than 50-fold compared to age-matched mammary glands from wildtype mice. Indeed, BAT has characteristics that are favorable for tumor growth, including high vascularity. Therefore, we also demonstrated that the multilocular brown adipose phenotype in the mammary fat pad of Brca1 mutant mice displayed regions of increased vascularity as evidenced by a significant increase in the protein expression of CD31, a marker for angiogenesis. This Brca1 mutant mouse model should provide a physiologically relevant context to determine whether brown adipose tissue can play a role in breast cancer development.

  2. Predominant neuronal B-cell loss in L5 DRG of p75 receptor-deficient mice

    PubMed Central

    Dreetz Gjerstad, M; Tandrup, T; Koltzenburg, M; Jakobsen, J

    2002-01-01

    The significance of the p75 low-affinity neurotrophin receptor, for the maintenance and survival of DRG cells, was studied in p75-deficient mice. Perikarya of the L5 DRG of 12-week-old p75 receptor-deficient mice and healthy Balb C mice were compared using stereological techniques. Following systematic sampling, the optical fractionator and the planar vertical rotator were used to estimate the number and mean volume of the cell bodies of the two neuronal subpopulations. The loss of B-cells was 57% (P < 0.00001), numbers being 7300 (CV = 0.12) in controls and 3100 in p75 receptor-deficient mice (CV = 0.18). Also, A-cells showed a significant loss of 39% (P < 0.0001), numbers being 2600 (CV = 0.12) in control mice and 1500 (CV = 0.16) in p75 receptor-deficient mice. The volume of A-cells was reduced by 30% (P < 0.01), from 24.700 µm3 (CV = 0.17) perikarya in p75 knock-out mice to 15.100 µm3 (CV = 0.17) in controls. B-cell volume did not change significantly. It is concluded that the p75 receptor plays a major role in the survival of DRG cells. The predominant loss of small B-cells indicates that the effect of neurotrophins is dependent upon the presence of the p75 low-affinity receptor. PMID:11833656

  3. Sunitinib DDI with paracetamol, diclofenac, mefenamic acid and ibuprofen shows sex-divergent effects on the tissue uptake and distribution pattern of sunitinib in mice.

    PubMed

    Tan, Siok Yean; Wong, Mei Mei; Tiew, Angela Lu Wun; Choo, Yai Wen; Lim, Suat Hun; Ooi, Ing Hong; Modamio, Pilar; Fernández, Cecilia; Mariño, Eduardo L; Segarra, Ignacio

    2016-10-01

    Pharmacokinetic interaction of sunitinib with diclofenac, paracetamol, mefenamic acid and ibuprofen was evaluated due to their P450 mediated metabolism and OATP1B1, OATP1B3, ABCB1, ABCG2 transporters overlapping features. Male and female mice were administered 6 sunitinib doses (60 mg/kg) PO every 12 h and 30 min before the last dose were administered vehicle (control groups), 250 mg/kg paracetamol, 30 mg/kg diclofenac, 50 mg/kg mefenamic acid or 30 mg/kg ibuprofen (study groups), euthanized 6 h post last administration and sunitinib plasma, liver, kidney, brain concentrations analyzed. Ibuprofen halved sunitinib plasma concentration in female mice (p < 0.01) and showed 59 % lower concentration than male mice (p < 0.05). Diclofenac and paracetamol female mice showed 45 and 25 % higher plasma concentrations than male mice which were 27 % lower in mefenamic acid female mice. Paracetamol increased 2.2 (p < 0.05) liver and 1.4-fold (p < 0.05) kidney sunitinib concentrations in male mice that were lower in female mice (p < 0.01, p < 0.001, respectively). Ibuprofen increased 2.9-fold (p < 0.01) liver concentration in male mice that were higher than in female mice (p < 0.001). Female control mice had 35 % higher sunitinib brain concentration than male mice but the concentration decreased 37, 33, 10 and 57 % in the diclofenac, paracetamol, mefenamic acid and ibuprofen (p < 0.001), respectively. Tissue-plasma concentrations correlations were nonsignificant in control, paracetamol, mefenamic acid and ibuprofen groups but was significant in the diclofenac group in male mice (liver, brain) and female mice (liver, kidney). These results portray gender-based sunitinib pharmacokinetic differences and NSAIDs selective effects on male or female mice, with potential clinical translatability.

  4. T cells from baxalpha transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA damage-induced cell death in the absence of p53.

    PubMed Central

    Brady, H J; Salomons, G S; Bobeldijk, R C; Berns, A J

    1996-01-01

    Baxalpha was isolated due to its interaction with Bcl-2. Baxalpha overexpression in an interleukin (IL)-3 dependent cell line accelerates apoptosis upon removal of the cytokine. The ratio of Baxalpha to Bcl-2 appears to be crucial for the effect. To study the action of the bax gene product in vivo, we have generated transgenic mice overexpressing Baxalpha specifically in T cells. Such T cells show accelerated apoptosis in response to gamma-radiation, dexamethasone and etoposide. By crossing baxalpha mice with bcl-2 transgenics we show that the critical nature of the Baxalpha:Bcl-2 ratio holds in primary T cells and that it can be manipulated to elicit a strong response to previously resisted stimuli. p53 has a role in the regulation of apoptosis in response to DNA-damaging agents. p53 directly activates transcription of the bax gene. The presence of the baxalpha transgene accelerated apoptosis in thymocytes from both p53-l- and p53+l- mice in response to dexamethasone. Thymocytes from p53-l- mice with the baxalpha transgene showed similar resistance to apoptosis by DNA-damaging agents as did p53-l- mice without the transgene. Baxalpha overexpression alone cannot restore the DNA damage apoptosis pathway, suggesting that p53 is required to induce or activate other factor(s) to reconstitute the response fully. Images PMID:8635454

  5. Fertility comparison between wild type and transgenic mice by in vitro fertilization.

    PubMed

    Vasudevan, Kuzhalini; Raber, James; Sztein, Jorge

    2010-08-01

    Transgenic mice are increasingly used as animal models for studies of gene function and regulation of mammalian genes. Although there has been continuous and remarkable progress in the development of transgenic technology over several decades, many aspects of the resulting transgenic model's phenotype cannot be completely predicted. For example, it is well known that as a consequence of the random insertion of the injected DNA construct, several founder mice of the new line need to be analyzed for possible differences in phenotype secondary to different insertion sites. The Knock out technique for transgenic production disrupts a specific gene by insertion or homologous recombination creating a null expression or replacement of the gene with a marker to localize it expression. This modification could result in pleiotropic phenotype if the gene is also expressed in tissues other than the target organs. Although the future breeding performance of the newly created model is critical to many studies, it is rarely anticipated that the new integrations could modify the reproductive profile of the new transgenic line. To date, few studies have demonstrated the difference between the parent strain's reproductive performance and the newly developed transgenic model. This study was designed to determine whether a genetic modification, knock out (KO) or transgenics, not anticipated to affect reproductive performance could affect the resulting reproductive profile of the newly developed transgenic mouse. More specifically, this study is designed to study the impact of the genetic modification on the ability of gametes to be fertilized in vitro. We analyzed the reproductive performance of mice with different background strains: FVB/N, C57BL/6 (129Sv/J x C57Bl/6)F1 and outbred CD1((R)) and compared them to mice of the same strain carrying a transgene or KO which was not anticipated to affect fertility. In vitro Fertilization was used to analyze the fertility of the mice. Oocytes

  6. Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice.

    PubMed

    Nicolussi, Simon; Viveros-Paredes, Juan Manuel; Gachet, María Salomé; Rau, Mark; Flores-Soto, Mario Eduardo; Blunder, Martina; Gertsch, Jürg

    2014-02-01

    High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50=290nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure-activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Help NCI at Frederick “Knock Out Hunger” | Poster

    Cancer.gov

    NCI at Frederick is once again participating in the Feds Feed Families initiative, an annual food drive that addresses severe shortages of non-perishable items in food banks across D.C., Maryland, and Virginia during the summer months, when giving is at its lowest.

  8. Moon formation: Punch combo or knock-out blow?

    NASA Astrophysics Data System (ADS)

    Collins, Gareth S.

    2017-01-01

    The twin isotopic signatures of the Moon and Earth are difficult to explain by a single giant impact. Impact simulations suggest that making the Moon by a combination of multiple, smaller moonlet-forming impacts may work better.

  9. Knocking out knotweed: research pins down a rogue invasive

    Treesearch

    Natasha Vizcarra; Shannon Claeson

    2015-01-01

    Bohemian knotweed spreads aggressively along rivers. This invasive weed chokes waterways, displaces native plants, erodes riverbanks, and keeps tree seedlings from growing. Communities in the Pacific Northwest spend millions of dollars to eradicate it on the assumption that it harms fish habitats.But knotweed is difficult to kill. It takes...

  10. ENU Mutagenesis Screen to Establish Motor Phenotypes in Wild-Type Mice and Modifiers of a Pre-Existing Motor Phenotype in Tau Mutant Mice

    PubMed Central

    Liu, Xin; Dobbie, Michael; Tunningley, Rob; Whittle, Belinda; Zhang, Yafei; Ittner, Lars M.; Götz, Jürgen

    2011-01-01

    Modifier screening is a powerful genetic tool. While not widely used in the vertebrate system, we applied these tools to transgenic mouse strains that recapitulate key aspects of Alzheimer's disease (AD), such as tau-expressing mice. These are characterized by a robust pathology including both motor and memory impairment. The phenotype can be modulated by ENU mutagenesis, which results in novel mutant mouse strains and allows identifying the underlying gene/mutation. Here we discuss this strategy in detail. We firstly obtained pedigrees that modify the tau-related motor phenotype, with mapping ongoing. We further obtained transgene-independent motor pedigrees: (i) hyperactive, circling ENU 37 mice with a causal mutation in the Tbx1 gene—the complete knock-out of Tbx1 models DiGeorge Syndrome; (ii) ENU12/301 mice that show sudden jerky movements and tremor constantly; they have a causal mutation in the Kcnq1 gene, modelling aspects of the Romano-Ward and Jervell and Lange-Nielsen syndromes; and (iii) ENU16/069 mice with tremor and hypermetric gait that have a causal mutation in the Mpz (Myelin Protein Zero) gene, modelling Charcot-Marie-Tooth disease type 1 (CMT1B). Together, we provide evidence for a real potential of an ENU mutagenesis to dissect motor functions in wild-type and tau mutant mice. PMID:22219655

  11. Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion*

    PubMed Central

    Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C.; Wang, Tong

    2016-01-01

    Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk−/− mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1−/− mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1+/+ and Romk1−/− mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1+/+, but such regulation by high K intake was diminished with significant hyperkalemia in Romk1−/− mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K+ secretion in the collecting tubule. PMID:26728465

  12. Conditional Knockout in Mice Reveals the Critical Roles of Ppp2ca in Epidermis Development.

    PubMed

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-05-18

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2ca(flox/flox) transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2ca(flox/flox); Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2ca(flox/flox); Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2ca(flox/flox) mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases.

  13. Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

    PubMed

    Krishnan, Shuba; Paredes, João A; Zhou, Xiaoshan; Kuiper, Raoul V; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-11-21

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2(-/-)) mice extended the life span of Tk2(-/-) mice from 3 weeks to at least 20 months. The Dm-dNK(+/-)Tk2(-/-) mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK(+/-)Tk2(-/-) mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK(+/-) mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.

  14. Transmembrane domain Nrg1 mutant mice show altered susceptibility to the neurobehavioural actions of repeated THC exposure in adolescence.

    PubMed

    Long, Leonora E; Chesworth, Rose; Huang, Xu-Feng; McGregor, Iain S; Arnold, Jonathon C; Karl, Tim

    2013-02-01

    Heavy cannabis abuse increases the risk of developing schizophrenia. Adolescents appear particularly vulnerable to the development of psychosis-like symptoms after cannabis use. To test whether the schizophrenia candidate gene neuregulin 1 (NRG1) modulates the effects of cannabinoids in adolescence, we tested male adolescent heterozygous transmembrane domain Nrg1 mutant (Nrg1 TM HET) mice and wild type-like littermates (WT) for their neurobehavioural response to repeated Δ(9)-tetrahydrocannabinol (THC, 10 mg/kg i.p. for 21 d starting on post-natal day 31). During treatment and 48 h after treatment withdrawal, we assessed several behavioural parameters relevant to schizophrenia. After behavioural testing we measured autoradiographic CB(1), 5-HT(2A) and NMDA receptor binding. The hyperlocomotor phenotype typical of Nrg1 mutants emerged after drug withdrawal and was more pronounced in vehicle than THC-treated Nrg1 TM HET mice. All mice were equally sensitive to THC-induced suppression of locomotion. However, mutant mice appeared protected against inhibiting effects of repeated THC on investigative social behaviours. Neither THC nor Nrg1 genotype altered prepulse inhibition. Repeated adolescent THC promoted differential effects on CB(1) and 5-HT(2A) receptor binding in the substantia nigra and insular cortex respectively, decreasing binding in WT while increasing it in Nrg1 TM HET mice. THC also selectively affected 5-HT(2A) receptor binding in several other regions in WT mice, whereas NMDA receptor binding was only affected in mutant mice. Overall, Nrg1 mutation does not appear to increase the induction of psychotomimetic symptoms by repeated adolescent THC exposure but may attenuate some of its actions on social behaviour and schizophrenia-relevant neurotransmitter receptor profiles.

  15. A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant.

    PubMed

    Mesquita, Leonardo Pereira; Gamon, Thais Helena Martins; Cuevas, Silvia Elena Campusano; Asano, Karen Miyuki; Fahl, Willian de Oliveira; Iamamoto, Keila; Scheffer, Karin Correa; Achkar, Samira Maria; Zanatto, Dennis Albert; Mori, Cláudia Madalena Cabrera; Maiorka, Paulo César; Mori, Enio

    2017-08-22

    Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.

  16. Lectin-like, oxidized low-density lipoprotein receptor-1-deficient mice show resistance to age-related knee osteoarthritis

    PubMed Central

    Hashimoto, Kazuhiko; Oda, Yutaka; Nakamura, Fumihisa; Kakinoki, Ryosuke; Akagi, Masao

    2017-01-01

    The lectin-like, oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1)/ox-LDL system contributes to atherosclerosis and may be involved in cartilage degeneration. The purpose of this study was to determine whether the LOX-1/ox-LDL system contributes to age-related osteoarthritis (OA) in vivo, using LOX-1 knockout (LOX-1 KO) mice. Knee cartilage from 6, 12, and 18-month old (n = 10/group) C57Bl/6 wild-type (WT) and LOX-1 KO mice was evaluated by determining the Osteoarthritis Research Society International (OARSI) score of Safranin-O stained samples. The prevalence of knee OA in both mouse strains was also investigated. Expression levels of LOX-1, ox-LDL, runt-related transcription factor-2 (Runx2), type-X collagen (COL X), and matrix metalloproteinase-13 (MMP-13) in the articular chondrocytes were analyzed immunohistologically. No significant difference was observed in the mean scores of WT (2.00±0.61) and LOX-1 KO mice (2.00±0.49) at 6 months of age (P=1.00, n=10). At 12 and 18 months of age, the mean scores of LOX-1 KO mice (3.75±0.93 and 5.50±0.78) were significantly lower than those of WT mice (5.25±1.14 and 9.00±1.01; P<0.001 in both cases; n=10). The prevalence of OA in LOX-1 KO mice was lower than that in WT mice at 12 and 18 months of age (40 vs 70%, 70 vs 90%, respectively; n=10). The expression levels of Runx2, COL X, and MMP-13 in articular chondrocytes significantly decreased in LOX-1 KO, mice compared with those in WT mice. The study indicated that the LOX-1/ox-LDL system in chondrocytes plays a role in the pathogenesis of age-related knee OA, which is potentially a target for preventing OA progression. PMID:28348422

  17. Elastin-insufficient mice show normal cardiovascular remodeling in 2K1C hypertension despite higher baseline pressure and unique cardiovascular architecture.

    PubMed

    Wagenseil, Jessica E; Knutsen, Russell H; Li, Dean Y; Mecham, Robert P

    2007-07-01

    Mice heterozygous for the elastin gene (ELN(+/-)) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN(+/-) mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN(+/-) cardiovascular system, we increased blood pressure in adult ELN(+/+) and ELN(+/-) mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN(+/+) and ELN(+/-)-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN(+/+) and ELN(+/-) hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN(+/-) mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.

  18. Transgenic mice overexpressing glia maturation factor-β, an oxidative stress inducible gene, show premature aging due to Zmpste24 down-regulation.

    PubMed

    Imai, Rika; Asai, Kanae; Hanai, Jun-ichi; Takenaka, Masaru

    2015-07-01

    Glia Maturation Factor-β (GMF), a brain specific protein, is induced by proteinuria in renal tubules. Ectopic GMF overexpression causes apoptosisin vitro via cellular vulnerability to oxidative stress. In order to examine the roles of GMF in non-brain tissue, we constructed transgenic mice overexpressing GMF (GMF-TG). The GMF-TG mice exhibited appearance phenotypes associated with premature aging. The GMF-TG mice also demonstrated short lifespans and reduced hair regrowth, suggesting an accelerated aging process. The production of an abnormal lamin A, a nuclear envelope protein, plays a causal role in both normal aging and accelerated aging diseases, known as laminopathies. Importantly, we identified the abnormal lamin A (prelamin A), accompanied by a down-regulation of a lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The GMF-TG mice showed accelerated aging in the kidney, compared with wild-type mice, showing increased TGF-β1, CTGF gene and serum creatinine. The gene expression of p21/waf1 was increased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at a later stage, at 60 weeks. In conclusion, we propose that GMF-TG mice might be a novel mouse model of accelerated aging, due to the abnormal lamin A.

  19. Flower-deficient mice have reduced susceptibility to skin papilloma formation

    PubMed Central

    Petrova, Evgeniya; López-Gay, Jesús M.; Rhiner, Christa; Moreno, Eduardo

    2012-01-01

    SUMMARY Skin papillomas arise as a result of clonal expansion of mutant cells. It has been proposed that the expansion of pretumoral cell clones is propelled not only by the increased proliferation capacity of mutant cells, but also by active cell selection. Previous studies in Drosophila describe a clonal selection process mediated by the Flower (Fwe) protein, whereby cells that express certain Fwe isoforms are recognized and forced to undergo apoptosis. It was further shown that knock down of fwe expression in Drosophila can prevent the clonal expansion of dMyc-overexpressing pretumoral cells. Here, we study the function of the single predicted mouse homolog of Drosophila Fwe, referred to as mFwe, by clonal overexpression of mFwe isoforms in Drosophila and by analyzing mFwe knock-out mice. We show that clonal overexpression of certain mFwe isoforms in Drosophila also triggers non-autonomous cell death, suggesting that Fwe function is evolutionarily conserved. Although mFwe-deficient mice display a normal phenotype, they develop a significantly lower number of skin papillomas upon exposure to DMBA/TPA two-stage skin carcinogenesis than do treated wild-type and mFwe heterozygous mice. Furthermore, mFwe expression is higher in papillomas and the papilloma-surrounding skin of treated wild-type mice compared with the skin of untreated wild-type mice. Thus, we propose that skin papilloma cells take advantage of mFwe activity to facilitate their clonal expansion. PMID:22362363

  20. Astrocytic β2 Adrenergic Receptor Gene Deletion Affects Memory in Aged Mice

    PubMed Central

    Jensen, Cathy Joanna; Demol, Frauke; Bauwens, Romy; Kooijman, Ron; Massie, Ann; Villers, Agnès; Ris, Laurence; De Keyser, Jacques

    2016-01-01

    In vitro and in vivo studies suggest that the astrocytic adrenergic signalling enhances glycogenolysis which provides energy to be transported to nearby cells and in the form of lactate. This energy source is important for motor and cognitive functioning. While it is suspected that the β2-adrenergic receptor on astrocytes might contribute to this energy balance, it has not yet been shown conclusively in vivo. Inducible astrocyte specific β2-adrenergic receptor knock-out mice were generated by crossing homozygous β2-adrenergic receptor floxed mice (Adrb2flox) and mice with heterozygous tamoxifen-inducible Cre recombinase-expression driven by the astrocyte specific L-glutamate/L-aspartate transporter promoter (GLAST-CreERT2). Assessments using the modified SHIRPA (SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment) test battery, swimming ability test, and accelerating rotarod test, performed at 1, 2 and 4 weeks, 6 and 12 months after tamoxifen (or vehicle) administration did not reveal any differences in physical health or motor functions between the knock-out mice and controls. However deficits were found in the cognitive ability of aged, but not young adult mice, reflected in impaired learning in the Morris Water Maze. Similarly, long-term potentiation (LTP) was impaired in hippocampal brain slices of aged knock-out mice maintained in low glucose media. Using microdialysis in cerebellar white matter we found no significant differences in extracellular lactate or glucose between the young adult knock-out mice and controls, although trends were detected. Our results suggest that β2-adrenergic receptor expression on astrocytes in mice may be important for maintaining cognitive health at advanced age, but is dispensable for motor function. PMID:27776147

  1. Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1

    PubMed Central

    Hebel, Tobias; Eisinger, Kristina; Neumeier, Markus; Rein-Fischboeck, Lisa; Pohl, Rebekka; Meier, Elisabeth M.; Boettcher, Alfred; Froehner, Stanley C.; Adams, Marvin E.; Liebisch, Gerhard; Krautbauer, Sabrina; Buechler, Christa

    2015-01-01

    The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2−/−) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2−/− mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2−/− mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2−/− mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity. PMID:25625330

  2. Ablation of neurons expressing melanin-concentrating hormone (MCH) in adult mice improves glucose tolerance independent of MCH signaling.

    PubMed

    Whiddon, Benjamin B; Palmiter, Richard D

    2013-01-30

    Melanin-concentrating hormone (MCH)-expressing neurons have been ascribed many roles based on studies of MCH-deficient mice. However, MCH neurons express other neurotransmitters, including GABA, nesfatin, and cocaine-amphetamine-regulated transcript. The importance of these other signaling molecules made by MCH neurons remains incompletely characterized. To determine the roles of MCH neurons in vivo, we targeted expression of the human diphtheria toxin receptor (DTR) to the gene for MCH (Pmch). Within 2 weeks of diphtheria toxin injection, heterozygous Pmch(DTR/+) mice lost 98% of their MCH neurons. These mice became lean but ate normally and were hyperactive, especially during a fast. They also responded abnormally to psychostimulants. For these phenotypes, ablation of MCH neurons recapitulated knock-out of MCH, so MCH appears to be the critical neuromodulator released by these neurons. In contrast, MCH-neuron-ablated mice showed improved glucose tolerance when compared with MCH-deficient mutant mice and wild-type mice. We conclude that MCH neurons regulate glucose tolerance through signaling molecules other than MCH.

  3. Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Blatner, Megan J.; Dang, An K.; Clevenger, Amy C.; Adams, Catherine E.; Restrepo, Diego

    2010-01-01

    Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expression (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knockout mice [α7−/−]) significantly differ in odor discrimination and detection of chemically related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics. PMID:20713028

  4. Differential effects of genes of the Rb1 signalling pathway on osteosarcoma incidence and latency in alpha-particle irradiated mice.

    PubMed

    Gonzalez-Vasconcellos, Iria; Domke, Tanja; Kuosaite, Virginija; Esposito, Irene; Sanli-Bonazzi, Bahar; Nathrath, Michaela; Atkinson, Michael J; Rosemann, Michael

    2011-03-01

    Osteosarcoma is the most frequent secondary malignancy following radiotherapy of patients with bilateral retinoblastoma. This suggests that the Rb1 tumour suppressor gene might confer genetic susceptibility towards radiation-induced osteosarcoma. To define the contribution of the Rb1 pathway in the multistep process of radiation carcinogenesis, we evaluated somatic allelic changes affecting the Rb1 gene itself as well as its upstream regulator p16 in murine osteosarcoma induced by (227)Th incorporation. To distinguish between the contribution of germline predisposition and the effect of a 2-hit allelic loss, two mouse models harbouring heterozygote germline Rb1 and p16 defects were tested for the incidence and latency of osteosarcoma following irradiation. We could show that all tumours arising in BALB/c×CBA/CA hybrid mice (wild-type for Rb1 and for p16) carried a somatic allelic loss of either the Rb1 gene (76.5%) or the p16 gene (59%). In none of the tumours, we found concordant retention of heterozygosity at both loci. Heterozygote knock-out mice for Rb1 exhibit a significant increase in the incidence of osteosarcoma following (227)Th incorporation (11/24 [corrected] in Rb1+/- vs. 2/18 in Rb1+/+, p=4×10(-5)), without affecting tumour latency. In contrast, heterozygote knock-out mice for p16 had no significant change in tumour incidence, but a pronounced reduction of latency (LT(50%) =355 days in p16+/- vs. 445 days in p16+/+, p=8×10(-3)). These data suggest that Rb1 germline defects influence early steps of radiation osteosarcomagenesis, whereas alterations in p16 mainly affect later stages of tumour promotion and growth.

  5. Forebrain-specific constitutively active CaMKKα transgenic mice show deficits in hippocampus-dependent long-term memory.

    PubMed

    Kaitsuka, Taku; Li, Sheng-Tian; Nakamura, Kenji; Takao, Keizo; Miyakawa, Tsuyoshi; Matsushita, Masayuki

    2011-09-01

    The Ca(2+)/calmodulin (CaM) kinase cascade is activated by Ca(2+) influx through the voltage-dependent Ca(2+) channels and the NMDA receptor. CaM kinase kinase (CaMKK), the most upstream kinase of the CaM kinase cascade, phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, resulting in activation of cyclic AMP-responsive element binding protein (CREB)-dependent gene transcription. Using transgenic techniques, we created mutant mice in which a constitutively active form of CaMKK1, the autoinhibitory domain truncated protein, is over-expressed specifically in the forebrain. In these mice, although performance was normal in basal activity and short-term memory, specific impairments were shown in hippocampus-dependent long-term memory after training in spatial memory tasks and after contextual fear conditioning. In cultured neurons of these mice, phosphorylation of CaMKI was significantly increased in basal states, whereas the activity range of CaMKI phosphorylation by brain-derived neurotrophic factor (BDNF) and KCl stimulation was significantly diminished in mutant mice. Our results define a critical role for CaMKKα in synaptic plasticity and the retention of hippocampus-dependent long-term memory.

  6. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

    PubMed

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

  7. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

    PubMed Central

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

  8. Deficiency of pantothenate kinase 2 (Pank2) in mice leads to retinal degeneration and azoospermia.

    PubMed

    Kuo, Yien-Ming; Duncan, Jacque L; Westaway, Shawn K; Yang, Haidong; Nune, George; Xu, Eugene Yujun; Hayflick, Susan J; Gitschier, Jane

    2005-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden-Spatz syndrome) is a rare but devastating neurodegenerative disorder, resulting from an inherited defect in coenzyme A biosynthesis. As pathology in the human condition is limited to the central nervous system, specifically the retina and globus pallidus, we have generated a mouse knock-out of the orthologous murine gene (Pank2) to enhance our understanding of the mechanisms of disease and to serve as a testing ground for therapies. Over time, the homozygous null mice manifest retinal degeneration, as evidenced by electroretinography, light microscopy and pupillometry response. Specifically, Pank2 mice show progressive photoreceptor decline, with significantly lower scotopic a- and b-wave amplitudes, decreased cell number and disruption of the outer segment and reduced pupillary constriction response when compared with those of wild-type littermates. Additionally, the homozygous male mutants are infertile due to azoospermia, a condition that was not appreciated in the human. Arrest occurs in spermiogenesis, with complete absence of elongated and mature spermatids. In contrast to the human, however, no changes were observed in the basal ganglia by MRI or by histological exam, nor were there signs of dystonia, even after following the mice for one year. Pank2 mice are 20% decreased in weight when compared with their wild-type littermates; however, dysphagia was not apparent. Immunohistochemistry shows staining consistent with localization of Pank2 to the mitochondria in both the retina and the spermatozoa.

  9. Strain-dependent differences in LTP and hippocampus-dependent memory in inbred mice.

    PubMed

    Nguyen, P V; Abel, T; Kandel, E R; Bourtchouladze, R

    2000-01-01

    Many studies have used "reverse" genetics to produce "knock-out" and transgenic mice to explore the roles of various molecules in long-term potentiation (LTP) and spatial memory. The existence of a variety of inbred strains of mice provides an additional way of exploring the genetic bases of learning and memory. We examined behavioral memory and LTP expression in area CA1 of hippocampal slices prepared from four different inbred strains of mice: C57BL/6J, CBA/J, DBA/2J, and 129/SvEms-+(Ter?)/J. We found that LTP induced by four 100-Hz trains of stimulation was robust and long-lasting in C57BL/6J and DBA/2J mice but decayed in CBA/J and 129/SvEms-+(Ter?)/J mice. LTP induced by one 100-Hz train was significantly smaller after 1 hr in the 129/SvEms-+(Ter?)/J mice than in the other three strains. Theta-burst LTP was shorter lasting in CBA/J, DBA/2J, and 129/SvEms-+(Ter?)/J mice than in C57BL/6J mice. We also observed specific memory deficits, among particular mouse strains, in spatial and nonspatial tests of hippocampus-dependent memory. CBA/J mice showed defective learning in the Morris water maze, and both DBA/2J and CBA/J strains displayed deficient long-term memory in contextual and cued fear conditioning tests. Our findings provide strong support for a genetic basis for some forms of synaptic plasticity that are linked to behavioral long-term memory and suggest that genetic background can influence the electrophysiological and behavioral phenotypes observed in genetically modified mice generated for elucidating the molecular bases of learning, memory, and LTP.

  10. 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.

  11. Vanin-1(-/-) mice show decreased NSAID- and Schistosoma-induced intestinal inflammation associated with higher glutathione stores.

    PubMed

    Martin, Florent; Penet, Marie-France; Malergue, Fabrice; Lepidi, Hubert; Dessein, Alain; Galland, Franck; de Reggi, Max; Naquet, Philippe; Gharib, Bouchra

    2004-02-01

    Vanin-1 is a membrane-anchored pantetheinase highly expressed in the gut and liver. It hydrolyzes pantetheine to pantothenic acid (vitamin B5) and the low-molecular-weight thiol cysteamine. The latter is believed to be a key regulating factor of several essential metabolic pathways, acting through sulfhydryl-disulfide exchange reactions between sulfhydryl groups of the enzymes and the oxidized form, cystamine. Its physiological importance remains to be elucidated, however. To explore this point, we developed Vanin-1-deficient mice that lack free cysteamine. We examined the susceptibility of deficient mice to intestinal inflammation, either acute (NSAID administration) or chronic (Schistosoma infection). We found that Vanin-1(-/-) mice better controlled inflammatory reaction and intestinal injury in both experiments. This protection was associated with increased gamma-glutamylcysteine synthetase activity and increased stores of reduced glutathione, as well as reduced inflammatory cell activation in inflamed tissues. Oral administration of cystamine reversed all aspects of the deficient phenotype. These findings suggest that one cysteamine function is to upregulate inflammation. Consequently, the pantetheinase activity of Vanin-1 molecule could be a target for a new anti-inflammatory strategy.

  12. Mice deficient in GEM GTPase show abnormal glucose homeostasis due to defects in beta-cell calcium handling.

    PubMed

    Gunton, Jenny E; Sisavanh, Mary; Stokes, Rebecca A; Satin, Jon; Satin, Leslie S; Zhang, Min; Liu, Sue M; Cai, Weikang; Cheng, Kim; Cooney, Gregory J; Laybutt, D Ross; So, Trina; Molero, Juan-Carlos; Grey, Shane T; Andres, Douglas A; Rolph, Michael S; Mackay, Charles R

    2012-01-01

    Glucose-stimulated insulin secretion from beta-cells is a tightly regulated process that requires calcium flux to trigger exocytosis of insulin-containing vesicles. Regulation of calcium handling in beta-cells remains incompletely understood. Gem, a member of the RGK (Rad/Gem/Kir) family regulates calcium channel handling in other cell types, and Gem over-expression inhibits insulin release in insulin-secreting Min6 cells. The aim of this study was to explore the role of Gem in insulin secretion. We hypothesised that Gem may regulate insulin secretion and thus affect glucose tolerance in vivo. Gem-deficient mice were generated and their metabolic phenotype characterised by in vivo testing of glucose tolerance, insulin tolerance and insulin secretion. Calcium flux was measured in isolated islets. Gem-deficient mice were glucose intolerant and had impaired glucose stimulated insulin secretion. Furthermore, the islets of Gem-deficient mice exhibited decreased free calcium responses to glucose and the calcium oscillations seen upon glucose stimulation were smaller in amplitude and had a reduced frequency. These results suggest that Gem plays an important role in normal beta-cell function by regulation of calcium signalling.

  13. Enzyme-treated Asparagus officinalis extract shows neuroprotective effects and attenuates cognitive impairment in senescence-accelerated mice.

    PubMed

    Sakurai, Takuya; Ito, Tomohiro; Wakame, Koji; Kitadate, Kentaro; Arai, Takashi; Ogasawara, Junetsu; Kizaki, Takako; Sato, Shogo; Ishibashi, Yoshinaga; Fujiwara, Tomonori; Akagawa, Kimio; Ishida, Hitoshi; Ohno, Hideki

    2014-01-01

    Increases in the number of patients with dementia involving Alzheimer's disease (AD) are seen as a grave public health problem. In neurodegenerative disorders involving AD, biological stresses, such as oxidative and inflammatory stress, induce neural cell damage. Asparagus (Asparagus officinalis) is a popular vegetable, and an extract prepared from this reportedly possesses various beneficial biological activities. In the present study, we investigated the effects of enzyme-treated asparagus extract (ETAS) on neuronal cells and early cognitive impairment of senescence-accelerated mouse prone 8 (SAMP8) mice. The expression of mRNAs for factors that exert cytoprotective and anti-apoptotic functions, such as heat-shock protein 70 and heme oxygenase-1, was upregulated in NG108-15 neuronal cells by treatment with ETAS. Moreover, when release of lactate dehydrogenase from damaged NG108-15 cells was increased for cells cultured in medium containing either the nitric oxide donor sodium nitroprusside or the hypoxia mimic reagent cobalt chloride, ETAS significantly attenuated this cell damage. Also, when contextual fear memory, which is considered to be a hippocampus-dependent memory, was significantly impaired in SAMP8 mice, ETAS attenuated the cognitive impairment. These results suggest that ETAS produces cytoprotective effects in neuronal cells and attenuates the effects on the cognitive impairment of SAMP8 mice.

  14. Mice Deficient in GEM GTPase Show Abnormal Glucose Homeostasis Due to Defects in Beta-Cell Calcium Handling

    PubMed Central

    Gunton, Jenny E.; Sisavanh, Mary; Stokes, Rebecca A.; Satin, Jon; Satin, Leslie S.; Zhang, Min; Liu, Sue M.; Cai, Weikang; Cheng, Kim; Cooney, Gregory J.; Laybutt, D. Ross; So, Trina; Molero, Juan-Carlos; Grey, Shane T.; Andres, Douglas A.

    2012-01-01

    Aims and Hypothesis Glucose-stimulated insulin secretion from beta-cells is a tightly regulated process that requires calcium flux to trigger exocytosis of insulin-containing vesicles. Regulation of calcium handling in beta-cells remains incompletely understood. Gem, a member of the RGK (Rad/Gem/Kir) family regulates calcium channel handling in other cell types, and Gem over-expression inhibits insulin release in insulin-secreting Min6 cells. The aim of this study was to explore the role of Gem in insulin secretion. We hypothesised that Gem may regulate insulin secretion and thus affect glucose tolerance in vivo. Methods Gem-deficient mice were generated and their metabolic phenotype characterised by in vivo testing of glucose tolerance, insulin tolerance and insulin secretion. Calcium flux was measured in isolated islets. Results Gem-deficient mice were glucose intolerant and had impaired glucose stimulated insulin secretion. Furthermore, the islets of Gem-deficient mice exhibited decreased free calcium responses to glucose and the calcium oscillations seen upon glucose stimulation were smaller in amplitude and had a reduced frequency. Conclusions These results suggest that Gem plays an important role in normal beta-cell function by regulation of calcium signalling. PMID:22761801

  15. Mice Over-Expressing the Myocardial Creatine Transporter Develop Progressive Heart Failure and Show Decreased Glycolytic Capacity

    PubMed Central

    Phillips, Darci; Hove, Michiel ten; Schneider, Jurgen E.; Wu, Colin O.; Sebag-Montefiore, Liam; Aponte, Angel M.; Lygate, Craig A.; Wallis, Julie; Clarke, Kieran; Watkins, Hugh; Balaban, Robert S.; Neubauer, Stefan

    2009-01-01

    The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32 week old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel 1H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the heart’s metabolic enzymes and identified 7 proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: α- and β-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59±0.02 μmol ATP produced/μg protein/min; CrT-OE, 0.31±0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102±3 μmol/g wet myocardium; CrT-OE, 78±13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the β-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study

  16. Experimental Chagas disease in Balb/c mice previously vaccinated with T. rangeli. II. The innate immune response shows immunological memory: reality or fiction?

    PubMed

    Basso, B; Marini, V

    2015-03-01

    Trypanosoma cruzi is a real challenge to the host's immune system, because it requires strong humoral and cellular immune response to remove circulating trypomastigote forms, and to prevent the replication of amastigote forms in tissues, involving many regulator and effector components. This protozoan is responsible for Chagas disease, a major public health problem in Latinamerica. We have developed a model of vaccination with Trypanosoma rangeli, a parasite closely related to T. cruzi, but nonpathogenic to humans, which reduces the infectiousness in three different species of animals, mice, dogs and guinea pigs, against challenge with T. cruzi. In a previous work, we demonstrated that mice vaccinated with T. rangeli showed important soluble mediators that stimulate phagocytic activity versus only infected groups. The aim of this work was to study the innate immune response in mice vaccinated or not with T. rangeli. Different population cells and some soluble mediators (cytokines) in peritoneal fluid and plasma in mice vaccinated-infected and only infected with T. cruzi were studied. In the first hours of challenge vaccinated mice showed an increase of macrophages, NK, granulocytes, and regulation of IL6, IFNγ, TNFα and IL10, with an increase of IL12, with respect to only infected mice. Furthermore an increase was observed of Li T, Li B responsible for adaptative response. Finally the findings showed that the innate immune response plays an important role in vaccinated mice for the early elimination of the parasites, complementary with the adaptative immune response, suggesting that vaccination with T. rangeli modulates the innate response, which develops some kind of immunological memory, recognizing shared antigens with T. cruzi. These results could contribute to the knowledge of new mechanisms which would have an important role in the immune response to Chagas disease.

  17. Mice lacking acid-sensing ion channels (ASIC) 1 or 2, but not ASIC3, show increased pain behaviour in the formalin test.

    PubMed

    Staniland, Amelia A; McMahon, Stephen B

    2009-07-01

    Extracellular acidification is a component of the inflammatory process and may be a factor driving the pain accompanying it. Acid-sensing ion channels (ASICs) are neuronal proton sensors and evidence suggests they are involved in signalling inflammatory pain. The aims of this study were to (1) clarify the role of ASICs in nociception and (2) confirm their involvement in inflammatory pain and determine whether this was subunit specific. This was achieved by (1) direct comparison of the sensitivity of ASIC1, ASIC2, ASIC3 and TRPV1 knockout mice versus wildtype littermates to acute thermal and mechanical noxious stimuli and (2) studying the behavioural responses of each transgenic strain to hind paw inflammation with either complete Freund's adjuvant (CFA) or formalin. Naïve ASIC1(-/-) and ASIC2(-/-) mice responded normally to acute noxious stimuli, whereas ASIC3(-/-) mice were hypersensitive to high intensity thermal stimuli. CFA injection decreased mechanical and thermal withdrawal thresholds for up to 8 days. ASIC2(-/-) mice had increased mechanical sensitivity on day 1 post-CFA compared to wildtype controls. TRPV1(-/-) mice had significantly reduced thermal, but not mechanical, hyperalgesia on all days after inflammation. Following formalin injection, ASIC1(-/-) and ASIC2(-/-), but not ASIC3(-/-) or TRPV1(-/-), mice showed enhanced pain behaviour, predominantly in the second phase of the test. These data suggest that whilst ASICs may play a role in mediating inflammatory pain, this role is likely to be modulatory and strongly dependent on channel subtype.

  18. γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer.

    PubMed

    Kober, Olivia I; Ahl, David; Pin, Carmen; Holm, Lena; Carding, Simon R; Juge, Nathalie

    2014-04-01

    Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ(-/-)) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ(-/-) mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ(-/-) mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ(-/-) mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ(-/-) and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.

  19. Factors secreted from dental pulp stem cells show multifaceted benefits for treating acute lung injury in mice.

    PubMed

    Wakayama, Hirotaka; Hashimoto, Naozumi; Matsushita, Yoshihiro; Matsubara, Kohki; Yamamoto, Noriyuki; Hasegawa, Yoshinori; Ueda, Minoru; Yamamoto, Akihito

    2015-08-01

    Acute respiratory distress syndrome (ARDS) is a severe inflammatory disorder characterized by acute respiratory failure, resulting from severe, destructive lung inflammation and irreversible lung fibrosis. We evaluated the use of stem cells derived from human exfoliated deciduous teeth (SHEDs) or SHED-derived serum-free conditioned medium (SHED-CM) as treatments for bleomycin (BLM)-induced mice acute lung injury (ALI), exhibiting several pathogenic features associated with the human disease ARDS. Mice with BLM-induced ALI with or without SHED or SHED-CM treatment were examined for weight loss and survival. The lung tissue was characterized by histological and real-time quantitative polymerase chain reaction analysis. The effects of SHED-CM on macrophage differentiation in vitro were also assessed. A single intravenous administration of either SHEDs or SHED-CM attenuated the lung injury and weight loss in BLM-treated mice and improved their survival rate. Similar recovery levels were seen in the SHEDs and SHED-CM treatment groups, suggesting that SHED improves ALI by paracrine mechanisms. SHED-CM contained multiple therapeutic factors involved in lung-regenerative mechanisms. Importantly, SHED-CM attenuated the BLM-induced pro-inflammatory response and generated an anti-inflammatory/tissue-regenerating environment, accompanied by the induction of anti-inflammatory M2-like lung macrophages. Furthermore, SHED-CM promoted the in vitro differentiation of bone marrow-derived macrophages into M2-like cells, which expressed high levels of Arginase1, CD206 and Ym-1. Our results suggest that SHED-secreted factors provide multifaceted therapeutic effects, including a strong M2-inducing activity, for treating BLM-induced ALI. This work may open new avenues for research on stem cell-based ARDS therapies. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  20. Sensory defects in Necdin deficient mice result from a loss of sensory neurons correlated within an increase of developmental programmed cell death

    PubMed Central

    Andrieu, David; Meziane, Hamid; Marly, Fabienne; Angelats, Corinne; Fernandez, Pierre-Alain; Muscatelli, Françoise

    2006-01-01

    Background The human NECDIN gene is involved in a neurodevelopmental disorder, Prader-Willi syndrome (PWS). Previously we reported a mouse Necdin knock-out model with similar defects to PWS patients. Despite the putative roles attributed to Necdin, mainly from in vitro studies, its in vivo function remains unclear. In this study, we investigate sensory-motor behaviour in Necdin deficient mice. We reveal cellular defects and analyse their cause. Results We report sensory differences in Necdin deficient mice compared to wild type animals. These differences led us to investigate sensory neuron development in Necdin deficient mouse embryos. First, we describe the expression pattern of Necdin in developing DRGs and report a reduction of one-third in specified sensory neurons in dorsal roots ganglia and show that this neuronal loss is achieved by E13.5, when DRGs sensory neurons are specified. In parallel, we observed an increase of 41% in neuronal apoptosis during the wave of naturally occurring cell death at E12.5. Since it is assumed that Necdin is a P75NTR interactor, we looked at the P75NTR-expressing cell population in Necdin knock-out embryos. Unexpectedly, Necdin loss of function has no effect on p75NTR expressing neurons suggesting no direct genetic interaction between Necdin and P75NTR in this context. Although we exclude a role of Necdin in axonal outgrowth from spinal sensory neurons in early developmental stages; such a role could occur later in neuronal differentiation. Finally we also exclude an anti-proliferative role of Necdin in developing sensory neurons. Conclusion Overall, our data show clearly that, in early development of the nervous system, Necdin is an anti-apoptotic or survival factor. PMID:17116257

  1. Heterozygous peripheral myelin protein 22-deficient mice are affected by a progressive demyelinating tomaculous neuropathy.

    PubMed

    Adlkofer, K; Frei, R; Neuberg, D H; Zielasek, J; Toyka, K V; Suter, U

    1997-06-15

    Hereditary neuropathy with liability to pressure palsy (HNPP) is associated with a heterozygous 1.5 megabase deletion on chromosome 17 that includes the peripheral myelin protein (PMP) gene PMP22. We show that heterozygous PMP22 knock-out mice, which carry only one functional pmp22 allele and thus genetically mimic HNPP closely, display similar morphological and electrophysiological features as observed in HNPP nerves. As reported previously, focal hypermyelinating structures called tomacula, the pathological hallmarks of HNPP, develop progressively in young PMP22(+/0) mice. By following the fate of tomacula during aging, we demonstrate now that these mutant animals are also interesting models for examining HNPP disease mechanisms. Subtle electrophysiological abnormalities are detected in PMP22(+/0) mice >1 year old, and a significant number of abnormally swollen and degenerating tomacula are present. Thinly myelinated axons and supernumerary Schwann cells forming onion bulbs as fingerprints of repeated cycles of demyelination and remyelination are also encountered frequently. Quantitative analyses using electron microscopy on cross sections and light microscopy on single teased nerve fibers suggest that tomacula are intrinsically unstable structures that are prone to degeneration; however, the severity of morphological and electrophysiological abnormalities in PMP22(+/0) mice is variable. These combined findings are reminiscent of the disease progression in HNPP and offer a possible explanation about why some HNPP patients develop a chronic motor and sensory neuropathy later in life that resembles demyelinating forms of Charcot-Marie-Tooth disease by both morphological and clinical criteria.

  2. Nerve injury induces robust allodynia and ectopic discharges in Nav1.3 null mutant mice

    PubMed Central

    Nassar, Mohammed A; Baker, Mark D; Levato, Alessandra; Ingram, Rachel; Mallucci, Giovanna; McMahon, Stephen B; Wood, John N

    2006-01-01

    Changes in sodium channel activity and neuronal hyperexcitability contribute to neuropathic pain, a major clinical problem. There is strong evidence that the re-expression of the embryonic voltage-gated sodium channel subunit Nav1.3 underlies neuronal hyperexcitability and neuropathic pain. Here we show that acute and inflammatory pain behaviour is unchanged in global Nav1.3 mutant mice. Surprisingly, neuropathic pain also developed normally in the Nav1.3 mutant mouse. To rule out any genetic compensation mechanisms that may have masked the phenotype, we investigated neuropathic pain in two conditional Nav1.3 mutant mouse lines. We used Nav1.8-Cre mice to delete Nav1.3 in nociceptors at E14 and NFH-Cre mice to delete Nav1.3 throughout the nervous system postnatally. Again normal levels of neuropathic pain developed after nerve injury in both lines. Furthermore, ectopic discharges from damaged nerves were unaffected by the absence of Nav1.3 in global knock-out mice. Our data demonstrate that Nav1.3 is neither necessary nor sufficient for the development of nerve-injury related pain. PMID:17052333

  3. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice.

    PubMed

    Seibenhener, Michael L; Wooten, Michael C

    2015-02-06

    Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

  4. Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

    PubMed Central

    Seibenhener, Michael L.; Wooten, Michael C.

    2015-01-01

    Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability. PMID:25742564

  5. Mice Homozygous for a Deletion in the Glaucoma Susceptibility Locus INK4 Show Increased Vulnerability of Retinal Ganglion Cells to Elevated Intraocular Pressure.

    PubMed

    Gao, Shan; Jakobs, Tatjana C

    2016-04-01

    A genomic region located on chromosome 9p21 is associated with primary open-angle glaucoma and normal tension glaucoma in genome-wide association studies. The genomic region contains the gene for a long noncoding RNA called CDKN2B-AS, two genes that code for cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/p16(INK4A) and CDKN2B/p15(INK4B)) and an additional protein (p14(ARF)). We used a transgenic mouse model in which 70 kb of murine chromosome 4, syntenic to human chromosome 9p21, are deleted to study whether this deletion leads to a discernible phenotype in ocular structures implicated in glaucoma. Homozygous mice of this strain were previously reported to show persistent hyperplastic primary vitreous. Fundus photography and optical coherence tomography confirmed that finding but showed no abnormalities for heterozygous mice. Optokinetic response, eletroretinogram, and histology indicated that the heterozygous and mutant retinas were normal functionally and morphologically, whereas glial cells were activated in the retina and optic nerve head of mutant eyes. In quantitative PCR, CDKN2B expression was reduced by approximately 50% in the heterozygous mice and by 90% in the homozygous mice, which suggested that the CDKN2B knock down had no deleterious consequences for the retina under normal conditions. However, compared with wild-type and heterozygous animals, the homozygous mice are more vulnerable to retinal ganglion cell loss in response to elevated intraocular pressure.

  6. Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function*

    PubMed Central

    Bancroft, Tara; Bouaouina, Mohamed; Roberts, Sophia; Lee, Monica; Calderwood, David A.; Schwartz, Martin; Simons, Michael; Sessa, William C.; Kyriakides, Themis R.

    2015-01-01

    Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair. PMID:25389299

  7. Experimental Chagas disease. Innate immune response in Balb/c mice previously vaccinated with Trypanosoma rangeli. I. The macrophage shows immunological memory: Reality or fiction?

    PubMed

    Basso, B; Marini, V

    2014-04-01

    Chagas' disease, caused by Trypanosoma cruzi, is a major vector borne health problem in Latin America and an emerging or re-emerging infectious disease in several countries. Immune response to T. cruzi infection is highly complex and involves many components, both regulators and effectors. Although different parasites have been shown to activate different mechanisms of innate immunity, T. cruzi is often able to survive and replicate in its host because they are well adapted to resisting host defences. An experimental model for vaccinating mice with Trypanosoma rangeli, a parasite closely related to T. cruzi, but nonpathogenic to humans, has been designed in our laboratory, showing protection against challenge with T. cruzi infection. The aim of this work was to analyze some mechanisms of the early innate immune response in T. rangeli vaccinated mice challenged with T. cruzi. For this purpose, some interactions were studied between T. cruzi and peritoneal macrophages of mice vaccinated with T. rangeli, infected or not with T. cruzi and the levels of some molecules or soluble mediators which could modify these interactions. The results in vaccinated animals showed a strong innate immune response, where the adherent cells of the vaccinated mice revealed important phagocytic activity, and some soluble mediator (Respiratory Burst: significantly increase, p ≤ 0.03; NO: the levels of vaccinated animals were lower than those of the control group; Arginasa: significantly increase, p ≤ 0.04). The results showed an important role in the early elimination of the parasites and their close relation with the absence of histological lesions that these animals present with regard to the only infected mice. This behaviour reveals that the macrophages act with some type of memory, recognizing the antigens to which they have previously been exposed, in mice were vaccinated with T. rangeli, which shares epitopes with T. cruzi. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Neuropeptide Y Overexpressing Female and Male Mice Show Divergent Metabolic but Not Gut Microbial Responses to Prenatal Metformin Exposure

    PubMed Central

    Salomäki-Myftari, Henriikka; Vähätalo, Laura H.; Ailanen, Liisa; Pietilä, Sami; Laiho, Asta; Hänninen, Arno; Pursiheimo, Juha-Pekka; Munukka, Eveliina; Rintala, Anniina; Savontaus, Eriika; Pesonen, Ullamari; Koulu, Markku

    2016-01-01

    Background Prenatal metformin exposure has been shown to improve the metabolic outcome in the offspring of high fat diet fed dams. However, if this is evident also in a genetic model of obesity and whether gut microbiota has a role, is not known. Methods The metabolic effects of prenatal metformin exposure were investigated in a genetic model of obesity, mice overexpressing neuropeptide Y in the sympathetic nervous system and in brain noradrenergic neurons (OE-NPYDβH). Metformin was given for 18 days to the mated female mice. Body weight, body composition, glucose tolerance and serum parameters of the offspring were investigated on regular diet from weaning and sequentially on western diet (at the age of 5–7 months). Gut microbiota composition was analysed by 16S rRNA sequencing at 10–11 weeks. Results In the male offspring, metformin exposure inhibited weight gain. Moreover, weight of white fat depots and serum insulin and lipids tended to be lower at 7 months. In contrast, in the female offspring, metformin exposure impaired glucose tolerance at 3 months, and subsequently increased body weight gain, fat mass and serum cholesterol. In the gut microbiota, a decline in Erysipelotrichaceae and Odoribacter was detected in the metformin exposed offspring. Furthermore, the abundance of Sutterella tended to be decreased and Parabacteroides increased. Gut microbiota composition of the metformin exposed male offspring correlated to their metabolic phenotype. Conclusion Prenatal metformin exposure caused divergent metabolic phenotypes in the female and male offspring. Nevertheless, gut microbiota of metformin exposed offspring was similarly modified in both genders. PMID:27681875

  9. Clock mutant mice with Jcl/ICR background shows an impaired learning ability in water maze, but not in passive avoidance, at the beginning of dark phase.

    PubMed

    Sei, Hiroyoshi; Oishi, Katsutaka; Sano, Atsuko; Seno, Hiromasa; Ohmori, Tetsuro; Morita, Yusuke; Ishida, Norio

    2006-06-01

    We observed the learning ability in Clock mutant mice with Jcl/ICR background (Clockj), a mice model of evening-type individuals, in the early part of dark phase. In order to estimate the learning ability, Morris water maze (WM) and passive avoidance (PA) test were performed. Release of acetylcholine, 5 hydroxytryptophan (5-HT) and dopamine (DA) in hippocampus was measured by in vivo microdialysis method. Clockj showed the impaired learning ability in the WM, but not in PA test. Hippocampal acetylcholine release was significantly attenuated in the Clockj in comparison to the wild-type mice. Neither 5-HT nor DA in the hippocampus was affected by the Clock mutation. Clock, an essential gene controlling circadian rhythm, may have an important role on the spatial learning and hippocampal cholinergic function, at least, at the beginning of the dark phase.

  10. Mandibular coronoid process in parathyroid hormone-related protein-deficient mice shows ectopic cartilage formation accompanied by abnormal bone modeling.

    PubMed

    Shibata, Shunichi; Suda, Naoto; Fukada, Kenji; Ohyama, Kimie; Yamashita, Yasuo; Hammond, Vicki E

    2003-07-01

    Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller, this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller, supporting previous immunohistochemical results.

  11. GABA type B receptor signaling in proopiomelanocortin neurons protects against obesity, insulin resistance, and hypothalamic inflammation in male mice on a high-fat diet.

    PubMed

    Ito, Yoshihiro; Banno, Ryoichi; Shibata, Miyuki; Adachi, Koichi; Hagimoto, Shigeru; Hagiwara, Daisuke; Ozawa, Yoshiharu; Goto, Motomitsu; Suga, Hidetaka; Sugimura, Yoshihisa; Bettler, Bernhard; Oiso, Yutaka; Arima, Hiroshi

    2013-10-23

    There is evidence suggesting that the GABA system in the arcuate nucleus, where orexigenic neuropeptide Y and agouti-related peptide as well as anorexigenic proopiomelanocortin (POMC) are expressed, plays an important role in energy balance. In this study, we generated POMC-specific GABAB receptor-deficient [knock-out (KO)] mice. Male KO mice on a high-fat diet (HFD) showed mild increases in body weight (BW) at the age of 9 weeks compared to wild-type (WT) mice, and the differences remained significant until 16 weeks old. However, there was no difference in BW in females between genotypes. While food intake was similar between genotypes, oxygen consumption was significantly decreased in the male KO mice. The insulin tolerance test revealed that the male KO mice were less insulin sensitive compared to WT mice at the age of 8 weeks, when there was no significant difference in BW between genotypes. Despite increased BW, POMC mRNA expression in the arcuate nucleus was significantly decreased in the KO mice compared to WT mice at the age of 16 weeks. Furthermore, the expression of TNFα as well as IL-6, proinflammatory markers in the hypothalamus, was significantly increased in the KO mice on a HFD compared to WT mice. This demonstrates that the deletion of GABAB receptors in POMC neurons in the male mice on a HFD results in obesity, insulin resistance, and hypothalamic inflammation. Furthermore, the decreased POMC expression in the obese KO mice suggests that the regulation of POMC expression through GABAB receptors is essential for proper energy balance.

  12. Anxious and nonanxious mice show similar hippocampal sensory evoked oscillations under urethane anesthesia: difference in the effect of buspirone.

    PubMed

    Horváth, János; Barkóczi, Balázs; Müller, Géza; Szegedi, Viktor

    2015-01-01

    Hippocampal oscillations recorded under urethane anesthesia are proposed to be modulated by anxiolytics. All classes of clinically effective anxiolytics were reported to decrease the frequency of urethane theta; however, recent findings raise concerns about the direct correlation of anxiolysis and the frequency of hippocampal theta. Here, we took advantage of our two inbred mouse strains displaying extremes of anxiety (anxious (AX) and nonanxious (nAX)) to compare the properties of hippocampal activity and to test the effect of an anxiolytic drugs. No difference was observed in the peak frequency or in the peak power between AX and nAX strains. Buspirone (Bus) applied in 2.5 mg/kg decreased anxiety of AX but did not have any effect on nAX as was tested by elevated plus maze and open field. Interestingly, Bus treatment increased hippocampal oscillatory frequency in the AX but left it unaltered in nAX mice. Saline injection did not have any effect on the oscillation. Paired-pulse facilitation was enhanced by Bus in the nAX, but not in the AX strain. Collectively, these results do not support the hypothesis that hippocampal activity under urethane may serve as a marker for potential anxiolytic drugs. Moreover, we could not confirm the decrease of frequency after anxiolytic treatment.

  13. Anxious and Nonanxious Mice Show Similar Hippocampal Sensory Evoked Oscillations under Urethane Anesthesia: Difference in the Effect of Buspirone

    PubMed Central

    Horváth, János; Barkóczi, Balázs; Müller,