Cardiovascular phenotype in Smad3 deficient mice with renovascular hypertension.

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Sonu Kashyap

Full Text Available Renovascular hypertension (RVH has deleterious effects on both the kidney and the heart. TGF-β signaling through Smad3 directs tissue fibrosis in chronic injury models. In the 2-kidney 1-clip (2K1C model of RVH, employing mice on the 129 genetic background, Smad3 deficiency (KO protects the stenotic kidney (STK from development of interstitial fibrosis. However, these mice have an increased incidence of sudden cardiac death following 2K1C surgery. The purpose of this study was to characterize the cardiovascular phenotype of these mice. Renal artery stenosis (RAS was established in Wild-type (WT and Smad3 KO mice (129 genetic background by placement of a polytetrafluoroethylene cuff on the right renal artery. Mortality was 25.5% for KO mice with RAS, 4.1% for KO sham mice, 1.2% for WT with RAS, and 1.8% for WT sham mice. Myocardial tissue of mice studied at 3 days following surgery showed extensive myocyte necrosis in KO but not WT mice. Myocyte necrosis was associated with a rapid induction of Ccl2 expression, macrophage influx, and increased MMP-9 activity. At later time points, both KO and WT mice developed myocardial fibrosis. No aortic aneurysms or dissections were observed at any time point. Smad3 KO mice were backcrossed to the C57BL/6J strain and subjected to RAS. Sudden death was observed at 10-14 days following surgery in 62.5% of mice; necropsy revealed aortic dissections as the cause of death. As observed in the 129 mice, the STK of Smad3 KO mice on the C57BL/6J background did not develop significant chronic renal damage. We conclude that the cardiovascular manifestations of Smad3 deficient mice are strain-specific, with myocyte necrosis in 129 mice and aortic rupture in C57BL/6J mice. Future studies will define mechanisms underlying this strain-specific effect on the cardiovascular system.

Narcolepsy susceptibility gene CCR3 modulates sleep-wake patterns in mice.

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Hiromi Toyoda

Full Text Available Narcolepsy is caused by the loss of hypocretin (Hcrt neurons and is associated with multiple genetic and environmental factors. Although abnormalities in immunity are suggested to be involved in the etiology of narcolepsy, no decisive mechanism has been established. We previously reported chemokine (C-C motif receptor 3 (CCR3 as a novel susceptibility gene for narcolepsy. To understand the role of CCR3 in the development of narcolepsy, we investigated sleep-wake patterns of Ccr3 knockout (KO mice. Ccr3 KO mice exhibited fragmented sleep patterns in the light phase, whereas the overall sleep structure in the dark phase did not differ between Ccr3 KO mice and wild-type (WT littermates. Intraperitoneal injection of lipopolysaccharide (LPS promoted wakefulness and suppressed both REM and NREM sleep in the light phase in both Ccr3 KO and WT mice. Conversely, LPS suppressed wakefulness and promoted NREM sleep in the dark phase in both genotypes. After LPS administration, the proportion of time spent in wakefulness was higher, and the proportion of time spent in NREM sleep was lower in Ccr3 KO compared to WT mice only in the light phase. LPS-induced changes in sleep patterns were larger in Ccr3 KO compared to WT mice. Furthermore, we quantified the number of Hcrt neurons and found that Ccr3 KO mice had fewer Hcrt neurons in the lateral hypothalamus compared to WT mice. We found abnormalities in sleep patterns in the resting phase and in the number of Hcrt neurons in Ccr3 KO mice. These observations suggest a role for CCR3 in sleep-wake regulation in narcolepsy patients.

PTX3 predicts severe disease in febrile patients at the emergency department

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de Kruif, Martijn D.; Limper, Maarten; Sierhuis, Karlien; Wagenaar, Jiri F. P.; Spek, C. Arnold; Garlanda, Cecilia; Cotena, Alessia; Mantovani, Alberto; ten Cate, Hugo; Reitsma, Pieter H.; van Gorp, Eric C. M.

2010-01-01

Objectives: The long pentraxin PTX3 is a promising marker of disease severity in severely ill patients. In order to identify patients warranting critical care as quickly as possible, we investigated the value of PTX3 as a biomarker for disease severity in patients presenting with fever at the

PTX3, a Humoral Pattern Recognition Molecule, in Innate Immunity, Tissue Repair, and Cancer.

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Garlanda, Cecilia; Bottazzi, Barbara; Magrini, Elena; Inforzato, Antonio; Mantovani, Alberto

2018-04-01

Innate immunity includes a cellular and a humoral arm. PTX3 is a fluid-phase pattern recognition molecule conserved in evolution which acts as a key component of humoral innate immunity in infections of fungal, bacterial, and viral origin. PTX3 binds conserved microbial structures and self-components under conditions of inflammation and activates effector functions (complement, phagocytosis). Moreover, it has a complex regulatory role in inflammation, such as ischemia/reperfusion injury and cancer-related inflammation, as well as in extracellular matrix organization and remodeling, with profound implications in physiology and pathology. Finally, PTX3 acts as an extrinsic oncosuppressor gene by taming tumor-promoting inflammation in murine and selected human tumors. Thus evidence suggests that PTX3 is a key homeostatic component at the crossroad of innate immunity, inflammation, tissue repair, and cancer. Dissecting the complexity of PTX3 pathophysiology and human genetics paves the way to diagnostic and therapeutic exploitation.

Functional consequences of brain glycogen deficiency on the sleep-wake cycle regulation in PTG-KO mice

KAUST Repository

Burlet-Godinot, S.

2017-12-31

Introduction: In the CNS, glycogen is mainly localized in astrocytes where its levels are linked to neuronal activity. Astrocytic glycogen synthesis is regulated by glycogen synthase (GS) activity that is positively controlled by protein targeting to glycogen (PTG) expression levels. Although the role of glycogen in sleep/wake regulation is still poorly understood, we have previously demonstrated that, following a 6 hour gentle sleep deprivation (GSD), PTG mRNA expression and GS activity increased in the brain in mice while glycogen levels were paradoxically maintained and not affected. In order to gain further insight on the role of PTG in this process, we studied the sleep/wake cycle parameters in PTG knockout (PTG-KO) mice under baseline conditions and after a 6 hour GSD. Glycogen levels as well as mRNAs expression of genes related to energy metabolism were also determined in several brain areas. Materials and methods: Adult male C57BL/6J (WT) and PTG-KO mice were sleep-recorded under baseline conditions (24 h recordings, 12 h light/dark cycle) and following 6 hours GSD from ZT00 to ZT06. Vigilance states were visually scored (4 s temporal window). Spectral analysis of the EEG signal was performed using a discrete Fourier transformation. Glycogen measurements and gene expression analysis were assessed using a biochemical assay and quantitative RT-PCR respectively, on separate cohorts in WT vs PTG-KO mice at the end of the 6 hours GSD or in control animals (CTL) in different brain structures. Results: Quantitative analysis of the sleep/wake cycle under baseline conditions did not reveal major differences between the WT and the PTG-KO mice. However, during the dark period, the PTG-KO mice showed a significant increase in the number of wake and slow wave sleep episodes (respectively +26.5±8% and +26.1±8%; p< 0.05) together with a significant shortening in their duration (-21.6±7.2% and -14.3±2.8%; p< 0.01). No such quantitative changes were observed during

Interactions of the humoral pattern recognition molecule PTX3 with the complement system

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Doni, Andrea; Garlanda, Cecilia; Bottazzi, Barbara

2012-01-01

The innate immune system comprises a cellular and a humoral arm. The long pentraxin PTX3 is a fluid phase pattern recognition molecule, which acts as an essential component of the humoral arm of innate immunity. PTX3 has antibody-like properties including interactions with complement components....... PTX3 interacts with C1q, ficolin-1 and ficolin-2 as well as mannose-binding lectin, recognition molecules in the classical and lectin complement pathways. The formation of these heterocomplexes results in cooperative pathogen recognition and complement activation. Interactions with C4b binding protein...

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

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Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Tissue inhibitor of metalloproteinase-3 knockout mice exhibit enhanced energy expenditure through thermogenesis.

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Yohsuke Hanaoka

Full Text Available Tissue inhibitors of metalloproteinases (TIMPs regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme, its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.

Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid–paclitaxel (CLA-PTX on B16-F10 melanoma

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Du R

2014-06-01

Full Text Available Ruo Du,1 Ting Zhong,1 Wei-Qiang Zhang,1 Ping Song,1 Wen-Ding Song,1 Yang Zhao,1 Chao-Wang,1 Yi-Qun Tang,3 Xuan Zhang,1,2 Qiang Zhang1,2 1Department of Pharmaceutics, 2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 3Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China Abstract: In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC-modified sterically stabilized liposomes (SSLs containing conjugated linoleic acid–paclitaxel (CLA-PTX. The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes’ cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 µg/g and 4 hours (1.17±0

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

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Wijayalath, Wathsala; Majji, Sai; Villasante, Eileen F; Brumeanu, Teodor D; Richie, Thomas L; Casares, Sofia

2014-09-30

Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates

Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

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Zhao Yarui

2011-01-01

Full Text Available Abstract Background Sphingomyelin synthase 2 (SMS2 contributes to de novo sphingomyelin (SM biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. Methods The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR and protein level examination (SMS activity assay. Result We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2 or GFP cDNA (AdV-GFP. On day six after intravenous infusion of 2 × 1011 particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p Conclusions Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or SAP trigger cross-activation of the complement system

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Ma, Ying Jie; Doni, Andrea; Skjødt, Mikkel-Ole

2011-01-01

The long pentraxin 3 (PTX3), serum amyloid P component (SAP) and C-reactive protein (CRP) belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via...... the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain, but not CRP. MBL:PTX3 complex formation resulted in recruitment of C......1q, but this was not seen for the MBL:SAP complex. However, both MBL:PTX3 and MBL:SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 lead to communication between the lectin and classical complement...

Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology.

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Dalla, C; Antoniou, K; Papadopoulou-Daifoti, Z; Balthazart, J; Bakker, J

2004-07-01

We recently found that female aromatase knockout (ArKO) mice that are deficient in oestradiol due to a targeted mutation in the aromatase gene show deficits in sexual behaviour that cannot be corrected by adult treatment with oestrogens. We determined here whether these impairments are associated with changes in general levels of activity, anxiety or 'depressive-like' symptomatology due to chronic oestrogen deficiency. We also compared the neurochemical profile of ArKO and wild-type (WT) females, as oestrogens have been shown to modulate dopaminergic, serotonergic and noradrenergic brain activities. ArKO females did not differ from WT in spontaneous motor activity, exploration or anxiety. These findings are in line with the absence of major neurochemical alterations in hypothalamus, prefrontal cortex or striatum, which are involved in the expression of these behaviours. By contrast, ArKO females displayed decreased active behaviours, such as struggling and swimming, and increased passive behaviours, such as floating, in repeated sessions of the forced swim test, indicating that these females exhibit 'depressive-like' symptoms. Adult treatment with oestradiol did not reverse the behavioural deficits observed in the forced swim test, suggesting that they may be due to the absence of oestradiol during development. Accordingly, an increased serotonergic activity was observed in the hippocampus of ArKO females compared with WT, which was also not reversed by adult oestradiol treatment. The possible organizational role of oestradiol on the hippocampal serotonergic system and the 'depressive-like' profile of ArKO females provide new insights into the pathophysiology of depression and the increased vulnerability of women to depression.

Hepatic metabolism affects the atropselective disposition of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in mice.

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Wu, Xianai; Barnhart, Christopher; Lein, Pamela J; Lehmler, Hans-Joachim

2015-01-06

To understand the role of hepatic vs extrahepatic metabolism in the disposition of chiral PCBs, we studied the disposition of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) and its hydroxylated metabolites (HO-PCBs) in mice with defective hepatic metabolism due to the liver-specific deletion of cytochrome P450 oxidoreductase (KO mice). Female KO and congenic wild type (WT) mice were treated with racemic PCB 136, and levels and chiral signatures of PCB 136 and HO-PCBs were determined in tissues and excreta 3 days after PCB administration. PCB 136 tissue levels were higher in KO compared to WT mice. Feces was a major route of PCB metabolite excretion, with 2,2',3,3',6,6'-hexachlorobiphenyl-5-ol being the major metabolite recovered from feces. (+)-PCB 136, the second eluting PCB 136 atropisomers, was enriched in all tissues and excreta. The second eluting atropisomers of the HO-PCBs metabolites were enriched in blood and liver; 2,2',3,3',6,6'-hexachlorobiphenyl-5-ol in blood was an exception and displayed an enrichment of the first eluting atropisomers. Fecal HO-PCB levels and chiral signatures changed with time and differed between KO and WT mice, with larger HO-PCB enantiomeric fractions in WT compared to KO mice. Our results demonstrate that hepatic and, possibly, extrahepatic cytochrome P450 (P450) enzymes play a role in the disposition of PCBs.

Isoproterenol induces vascular oxidative stress and endothelial dysfunction via a Giα-coupled β2-adrenoceptor signaling pathway.

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Ana P Davel

Full Text Available OBJECTIVE: Sustained β-adrenergic stimulation is a hallmark of sympathetic hyperactivity in cardiovascular diseases. It is associated with oxidative stress and altered vasoconstrictor tone. This study investigated the β-adrenoceptor subtype and the signaling pathways implicated in the vascular effects of β-adrenoceptor overactivation. METHODS AND RESULTS: Mice lacking the β1- or β2-adrenoceptor subtype (β1KO, β2KO and wild-type (WT were treated with isoproterenol (ISO, 15 μg.g(-1 x day(-1, 7 days. ISO significantly enhanced the maximal vasoconstrictor response (Emax of the aorta to phenylephrine in WT (+34% and β1KO mice (+35% but not in β2KO mice. The nitric oxide synthase (NOS inhibitor L-NAME abolished the differences in phenylephrine response between the groups, suggesting that ISO impaired basal NO availability in the aorta of WT and β1KO mice. Superoxide dismutase (SOD, pertussis toxin (PTx or PD 98,059 (p-ERK 1/2 inhibitor incubation reversed the hypercontractility of aortic rings from ISO-treated WT mice; aortic contraction of ISO-treated β2KO mice was not altered. Immunoblotting revealed increased aortic expression of Giα-3 protein (+50% and phosphorylated ERK1/2 (+90% and decreased eNOS dimer/monomer ratio in ISO-treated WT mice. ISO enhanced the fluorescence response to dihydroethidium (+100% in aortas from WT mice, indicating oxidative stress that was normalized by SOD, PTx and L-NAME. The ISO effects were abolished in β2KO mice. CONCLUSIONS: The β2-adrenoceptor/Giα signaling pathway is implicated in the enhanced vasoconstrictor response and eNOS uncoupling-mediated oxidative stress due to ISO treatment. Thus, long-term β2-AR activation might results in endothelial dysfunction.

Functional consequences of brain glycogen deficiency on the sleep-wake cycle regulation in PTG-KO mice

KAUST Repository

Burlet-Godinot, S.; Allaman, I.; Grenningloh, G.; Roach, P.J.; Depaoli-Roach, A.A.; Magistretti, Pierre J.; Petit, J.-M.

2017-01-01

in the brain in mice while glycogen levels were paradoxically maintained and not affected. In order to gain further insight on the role of PTG in this process, we studied the sleep/wake cycle parameters in PTG knockout (PTG-KO) mice under baseline conditions

Both chronic treatments by epothilone D and fluoxetine increase the short-term memory and differentially alter the mood status of STOP/MAP6 KO mice.

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Fournet, Vincent; de Lavilléon, Gaetan; Schweitzer, Annie; Giros, Bruno; Andrieux, Annie; Martres, Marie-Pascale

2012-12-01

Recent evidence underlines the crucial role of neuronal cytoskeleton in the pathophysiology of psychiatric diseases. In this line, the deletion of STOP/MAP6 (Stable Tubule Only Polypeptide), a microtubule-stabilizing protein, triggers various neurotransmission and behavioral defects, suggesting that STOP knockout (KO) mice could be a relevant experimental model for schizoaffective symptoms. To establish the predictive validity of such a mouse line, in which the brain serotonergic tone is dramatically imbalanced, the effects of a chronic fluoxetine treatment on the mood status of STOP KO mice were characterized. Moreover, we determined the impact, on mood, of a chronic treatment by epothilone D, a taxol-like microtubule-stabilizing compound that has previously been shown to improve the synaptic plasticity deficits of STOP KO mice. We demonstrated that chronic fluoxetine was either antidepressive and anxiolytic, or pro-depressive and anxiogenic, depending on the paradigm used to test treated mutant mice. Furthermore, control-treated STOP KO mice exhibited paradoxical behaviors, compared with their clear-cut basal mood status. Paradoxical fluoxetine effects and control-treated STOP KO behaviors could be because of their hyper-reactivity to acute and chronic stress. Interestingly, both epothilone D and fluoxetine chronic treatments improved the short-term memory of STOP KO mice. Such treatments did not affect the serotonin and norepinephrine transporter densities in cerebral areas of mice. Altogether, these data demonstrated that STOP KO mice could represent a useful model to study the relationship between cytoskeleton, mood, and stress, and to test innovative mood treatments, such as microtubule-stabilizing compounds. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

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Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Kim, Jin-Hoi

2018-02-27

The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice

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Abu Musa Md Talimur Reza

2018-02-01

Full Text Available The Rag2 knockout (KO mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs and messenger RNAs (mRNAs in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

Relaxin-3 receptor (RXFP3 signalling mediates stress-related alcohol preference in mice.

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Andrew W Walker

Full Text Available Stressful life events are causally linked with alcohol use disorders (AUDs, providing support for a hypothesis that alcohol consumption is aimed at stress reduction. We have previously shown that expression of relaxin-3 mRNA in rat brain correlates with alcohol intake and that central antagonism of relaxin-3 receptors (RXFP3 prevents stress-induced reinstatement of alcohol-seeking. Therefore the objectives of these studies were to investigate the impact of Rxfp3 gene deletion in C57BL/6J mice on baseline and stress-related alcohol consumption. Male wild-type (WT and Rxfp3 knockout (KO (C57/B6JRXFP3TM1/DGen littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in mice following chronic alcohol intake and in naive controls. WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and hepatic alcohol processing. However, Rxfp3 KO mice displayed a stress-induced reduction in alcohol preference that was not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing effects of alcohol.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

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Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

The sweet side of a long pentraxin: how glycosylation affects PTX3 functions in innate immunity and inflammation

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Antonio eInforzato

2013-01-01

Full Text Available Innate immunity represents the first line of defence against pathogens and plays key roles in activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules (PRMs that recognise pathogen associated molecular patterns (PAMPs and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. The long pentraxin PTX3 is a prototypic soluble PRM that is produced at sites of infection and inflammation by both somatic and immune cells. Gene targeting of this evolutionarily conserved protein has revealed a non-redundant role in resistance to selected pathogens. Moreover, PTX3 exerts important functions at the crossroad between innate immunity, inflammation and female fertility. The human PTX3 protein contains a single N-glycosylation site that is fully occupied by complex type oligosaccharides, mainly fucosylated and sialylated biantennary glycans. Glycosylation has been implicated in a number of PTX3 activities, including neutralization of influenza viruses, modulation of the complement system, and attenuation of leukocyte recruitment. Therefore, this post translational modification might act as a fine tuner of PTX3 functions in native immunity and inflammation.Here we review the studies on PTX3, with emphasis on the glycan-dependent mechanisms underlying pathogen recognition and crosstalk with other components of the innate immune system.

Ficolin-1-PTX3 complex formation promotes clearance of altered self-cells and modulates IL-8 production

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Ma, Ying Jie; Doni, Andrea; Romani, Luigina

2013-01-01

for enhancement of phagocytosis by human monocyte-derived macrophages and downregulation of IL-8 production during phagocytosis. On A. fumigatus, PTX3 exposed the C-terminal portion of the molecule, probably resulting in steric hindrance of ficolin-1 interaction with PTX3. These results demonstrate that ficolin-1...

PTX3 is up-regulated in epithelial mammary cells during S. aureus intramammary infection in goat

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Joel Fernando Soares Filipe

2015-07-01

PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, demonstrating that it represents a first line of immune defense in goat udder. No modulation was observed in macrophages, in the secretum and in the ductal epithelial cells. Further experiments are needed to elucidate the role of PTX3 in the pathogenesis of S. aureus infection.

Hepatic Metabolism Affects the Atropselective Disposition of 2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) in Mice

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2015-01-01

To understand the role of hepatic vs extrahepatic metabolism in the disposition of chiral PCBs, we studied the disposition of 2,2′,3,3′,6,6′-hexachlorobiphenyl (PCB 136) and its hydroxylated metabolites (HO-PCBs) in mice with defective hepatic metabolism due to the liver-specific deletion of cytochrome P450 oxidoreductase (KO mice). Female KO and congenic wild type (WT) mice were treated with racemic PCB 136, and levels and chiral signatures of PCB 136 and HO-PCBs were determined in tissues and excreta 3 days after PCB administration. PCB 136 tissue levels were higher in KO compared to WT mice. Feces was a major route of PCB metabolite excretion, with 2,2′,3,3′,6,6′-hexachlorobiphenyl-5-ol being the major metabolite recovered from feces. (+)-PCB 136, the second eluting PCB 136 atropisomers, was enriched in all tissues and excreta. The second eluting atropisomers of the HO-PCBs metabolites were enriched in blood and liver; 2,2′,3,3′,6,6′-hexachlorobiphenyl-5-ol in blood was an exception and displayed an enrichment of the first eluting atropisomers. Fecal HO-PCB levels and chiral signatures changed with time and differed between KO and WT mice, with larger HO-PCB enantiomeric fractions in WT compared to KO mice. Our results demonstrate that hepatic and, possibly, extrahepatic cytochrome P450 (P450) enzymes play a role in the disposition of PCBs. PMID:25420130

Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice

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Nørregaard, Rikke; Tao, Shixin; Nilsson, Line

2015-01-01

vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3αKO mice, suggesting......KO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3α could play a crucial role in renal urine concentration and suggest that GSK3α might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus....

Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish.

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Gros-Louis, Francois; Kriz, Jasna; Kabashi, Edor; McDearmid, Jonathan; Millecamps, Stéphanie; Urushitani, Makoto; Lin, Li; Dion, Patrick; Zhu, Qinzhang; Drapeau, Pierre; Julien, Jean-Pierre; Rouleau, Guy A

2008-09-01

Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes

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2014-01-01

Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice. PMID:24758191

Endogenous murine tau promotes neurofibrillary tangles in 3xTg-AD mice without affecting cognition.

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Baglietto-Vargas, David; Kitazawa, Masashi; Le, Elaine J; Estrada-Hernandez, Tatiana; Rodriguez-Ortiz, Carlos J; Medeiros, Rodrigo; Green, Kim N; LaFerla, Frank M

2014-02-01

Recent studies on tauopathy animal models suggest that the concomitant expression of the endogenous murine tau delays the pathological accumulation of human tau, and interferes with the disease progression. To elucidate the role of endogenous murine tau in a model with both plaques and tangles, we developed a novel transgenic mouse model by crossing 3xTg-AD with mtauKO mice (referred to as 3xTg-AD/mtauKO mice). Therefore, this new model allows us to determine the pathological consequences of the murine tau. Here, we show that 3xTg-AD/mtauKO mice have lower tau loads in both soluble and insoluble fractions, and lower tau hyperphosphorylation level in the soluble fraction relative to 3xTg-AD mice. In the 3xTg-AD model endogenous mouse tau is hyperphosphorylated and significantly co-aggregates with human tau. Despite the deletion of the endogenous tau gene in 3xTg-AD/mtauKO mice, cognitive dysfunction was equivalent to 3xTg-AD mice, as there was no additional impairment on a spatial memory task, and thus despite increased tau phosphorylation, accumulation and NFTs in 3xTg-AD mice no further effects on cognition are seen. These findings provide better understanding about the role of endogenous tau to Alzheimer's disease (AD) pathology and for developing new AD models. © 2013.

Severe systemic toxicity and urinary bladder cytotoxicity and regenerative hyperplasia induced by arsenite in arsenic (+ 3 oxidation state) methyltransferase knockout mice. A preliminary report

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Yokohira, Masanao; Arnold, Lora L.; Pennington, Karen L.; Suzuki, Shugo; Kakiuchi-Kiyota, Satoko; Herbin-Davis, Karen; Thomas, David J.; Cohen, Samuel M.

2010-01-01

Arsenic (+ 3 oxidation state) methyltransferase (As3mt) catalyzes reactions which convert inorganic arsenic to methylated metabolites. This study determined whether the As3mt null genotype in the mouse modifies cytotoxic and proliferative effects seen in urinary bladders of wild type mice after exposure to inorganic arsenic. Female wild type C57BL/6 mice and As3mt KO mice were divided into 3 groups each (n = 8) with free access to a diet containing 0, 100 or 150 ppm of arsenic as arsenite (As III ). During the first week of As III exposure, As3mt KO mice exhibited severe and lethal systemic toxicity. At termination, urinary bladders of both As3mt KO and wild type mice showed hyperplasia by light microscopy. As expected, arsenic-containing granules were found in the superficial urothelial layer of wild type mice. In As3mt KO mice these granules were present in all layers of the bladder epithelium and were more abundant and larger than in wild type mice. Scanning electron microscopy of the bladder urothelium of As3mt KO mice treated with 100 ppm As III showed extensive superficial necrosis and hyperplastic changes. In As3mt KO mice, livers showed severe acute inflammatory changes and spleen size and lymphoid areas were decreased compared with wild type mice. Thus, diminished arsenic methylation in As3mt KO mice exacerbates systemic toxicity and the effects of As III on the bladder epithelium, showing that altered kinetic and dynamic behavior of arsenic can affect its toxicity.

Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: Effect of sex and arsenic exposure

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Huang, Madelyn C.; Douillet, Christelle; Su, Mingming; Zhou, Kejun; Wu, Tao; Chen, Wenlian; Galanko, Joseph A.; Drobná, Zuzana; Saunders, R. Jesse; Martin, Elizabeth; Fry, Rebecca C.; Jia, Wei; Stýblo, Miroslav

2016-01-01

Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex-specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation. PMID:26883664

Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice

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Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R.; Kakade, Vijayakumar; Yu, Alan S. L.; Howard, Christiana; Rao, Reena

2015-01-01

In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine outp...

Effect of pertussis and cholera toxins administered supraspinally on CA3 hippocampal neuronal cell death and the blood glucose level induced by kainic acid in mice.

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Kim, Chea-Ha; Park, Soo-Hyun; Sim, Yun-Beom; Sharma, Naveen; Kim, Sung-Su; Lim, Su-Min; Jung, Jun-Sub; Suh, Hong-Won

2014-12-01

The effect of cholera toxin (CTX) or pertussis toxin (PTX) administered supraspinally on hippocampal neuronal cell death in CA3 region induced by kainic acid (KA) was examined in mice. After the pretreatment with either PTX or CTX intracerebroventricularly (i.c.v.), mice were administered i.c.v. with KA. The i.c.v. treatment with KA caused a neuronal cell death in CA3 region and PTX, but not CTX, attenuated the KA-induced neuronal cell death. In addition, i.c.v. treatment with KA caused an elevation of the blood glucose level. The i.c.v. PTX pretreatment alone caused a hypoglycemia and inhibited KA-induced hyperglycemic effect. However, i.c.v. pretreatment with CTX did not affect the basal blood glucose level and KA-induced hyperglycemic effect. Moreover, KA administered i.c.v. caused an elevation of corticosterone level and reduction of the blood insulin level. Whereas, i.c.v. pretreatment with PTX further enhanced KA-induced up-regulation of corticosterone level. Furthermore, i.c.v. administration of PTX alone increased the insulin level and KA-induced hypoinsulinemic effect was reversed. In addition, PTX pretreatment reduces the KA-induced seizure activity. Our results suggest that supraspinally administered PTX, exerts neuroprotective effect against KA-induced neuronal cells death in CA3 region and neuroprotective effect of PTX is mediated by the reduction of KA-induced blood glucose level. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

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Ilic, Zoran; Crawford, Dana; Egner, Patricia A.; Sell, Stewart

2010-01-01

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N 7 -DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N 7 -DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

Mitogen-activated protein kinase phosphatase-3 (MKP-3 in the surgical wound is necessary for the resolution of postoperative pain in mice

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Skopelja-Gardner S

2017-03-01

Full Text Available Sladjana Skopelja-Gardner,1,* Madhurima Saha,1,* Perla Abigail Alvarado-Vazquez,2 Brenna S Liponis,1 Elena Martinez,1 E Alfonso Romero-Sandoval2 1Department of Anesthesiology, Geisel School of Medicine at Dartmouth, Lebanon, NH, 2Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of Pharmacy, Clinton, SC, USA *These authors contributed equally to this work. Abstract: Mitogen-activated protein kinase (MAPK phosphatase-3 (MKP-3 and its substrates (extracellular signal-regulated kinase [ERK] and p38 play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT and MKP-3 knockout (KO mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2 inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08 g day 1, which resolved normally by postoperative day 12 (0.46±0.08 g, N=6. In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6. KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm2 than WT mice (8.7±1.2 cells/0.1 mm2 on postoperative day 1 (vs 5–6 cells/0.1 mm2 at baseline that returned to baseline 12 days after surgery (10–12 cells/0.1 mm2. In WT mice, peripheral p-p38 and p-ERK-1/2 expression increased (5- and 3-fold, respectively on postoperative days 1 and 5, and returned to basal levels 7–12 days after surgery (N=3 per group. Peripheral p-p38 levels in MKP-3 KO mice followed

Genes for carbon metabolism and the ToxA virulence factor in Pseudomonas aeruginosa are regulated through molecular interactions of PtxR and PtxS.

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Abdelali Daddaoua

Full Text Available Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the P(gad and P(kgu for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of Pseudomonas aeruginosa. We show using DNaseI-footprint analysis that in P. aeruginosa PtxR binds to the -35 region of the P(toxA promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the P(kgu and P(gad promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. In vitro, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of P(kgu and P(gad promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the P(toxA promoter, or via the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator.

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

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Ken Murakami

Full Text Available 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.

Magnetic properties of the alkali metal ozonides KO3, RbO3, and CsO3

International Nuclear Information System (INIS)

Lueken, H.; Deussen, M.; Jansen, M.; Hesse, W.; Schnick, W.

1987-01-01

The magnetic susceptibilities of KO 3 , RbO 3 and CsO 3 have been determined between 3.6 and 250 K. Above 50 K Curie-Weiss behaviour is observed. Magnetic moments of 1.74 μ B (KO 3 , CsO 3 ) and 1.80 μ B (RbO 3 ) calculated from the Curie-Weiss straight lines correspond with spin-only moments expected for isolated O 3 - species with one unpaired electron. The Weiss constants Θ are -34 K (KO 3 ), -23 K (RbO 3 ) and -10 K (CsO 3 ). The low temperature behaviour of KO 3 and RbO 3 (broad maxima in susceptibility at 20 and 17 K, respectively, and minima at 6 K) is typical of systems which show with decreasing temperature low-dimensional antiferromagnetic and three-dimensional magnetic ordering. Inspecting the intermolecular distances between oxygen atoms the pathways of exchange interactions are discussed. (author)

Sensitivity to Chronic Methamphetamine Administration and Withdrawal in Mice with Relaxin-3/RXFP3 Deficiency.

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Haidar, Mouna; Lam, Monica; Chua, Berenice E; Smith, Craig M; Gundlach, Andrew L

2016-03-01

Methamphetamine (METH) is a highly addictive psychostimulant, and cessation of use is associated with reduced monoamine signalling, and increased anxiety/depressive states. Neurons expressing the neuropeptide, relaxin-3 (RLN3), and its cognate receptor, RXFP3, constitute a putative 'ascending arousal system', which shares neuroanatomical and functional similarities with serotonin (5-HT)/dorsal raphe and noradrenaline (NA)/locus coeruleus monoamine systems. In light of possible synergistic roles of RLN3 and 5-HT/NA, endogenous RLN3/RXFP3 signalling may compensate for the temporary reduction in monoamine signalling associated with chronic METH withdrawal, which could alter the profile of 'behavioural despair', bodyweight reductions, and increases in anhedonia and anxiety-like behaviours observed following chronic METH administration. In studies to test this theory, Rln3 and Rxfp3 knockout (KO) mice and their wildtype (WT) littermates were injected once daily with saline or escalating doses of METH (2 mg/kg, i.p. on day 1, 4 mg/kg, i.p. on day 2 and 6 mg/kg, i.p. on day 3-10). WT and Rln3 and Rxfp3 KO mice displayed an equivalent sensitivity to behavioural despair (Porsolt swim) during the 2-day METH withdrawal and similar bodyweight reductions on day 3 of METH treatment. Furthermore, during a 3-week period after the cessation of chronic METH exposure, Rln3 KO, Rxfp3 KO and corresponding WT mice displayed similar behavioural responses in paradigms that measured anxiety (light/dark box, elevated plus maze), anhedonia (saccharin preference), and social interaction. These findings indicate that a whole-of-life deficiency in endogenous RLN3/RXFP3 signalling does not markedly alter behavioural sensitivity to chronic METH treatment or withdrawal, but leave open the possibility of a more significant interaction with global or localised manipulations of this peptide system in the adult brain.

Region-Specific Defects of Respiratory Capacities in the Ndufs4(KO Mouse Brain.

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Ernst-Bernhard Kayser

Full Text Available Lack of NDUFS4, a subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase, causes Leigh syndrome (LS, a progressive encephalomyopathy. Knocking out Ndufs4, either systemically or in brain only, elicits LS in mice. In patients as well as in KO mice distinct regions of the brain degenerate while surrounding tissue survives despite systemic complex I dysfunction. For the understanding of disease etiology and ultimately for the development of rationale treatments for LS, it appears important to uncover the mechanisms that govern focal neurodegeneration.Here we used the Ndufs4(KO mouse to investigate whether regional and temporal differences in respiratory capacity of the brain could be correlated with neurodegeneration. In the KO the respiratory capacity of synaptosomes from the degeneration prone regions olfactory bulb, brainstem and cerebellum was significantly decreased. The difference was measurable even before the onset of neurological symptoms. Furthermore, neither compensating nor exacerbating changes in glycolytic capacity of the synaptosomes were found. By contrast, the KO retained near normal levels of synaptosomal respiration in the degeneration-resistant/resilient "rest" of the brain. We also investigated non-synaptic mitochondria. The KO expectedly had diminished capacity for oxidative phosphorylation (state 3 respiration with complex I dependent substrate combinations pyruvate/malate and glutamate/malate but surprisingly had normal activity with α-ketoglutarate/malate. No correlation between oxidative phosphorylation (pyruvate/malate driven state 3 respiration and neurodegeneration was found: Notably, state 3 remained constant in the KO while in controls it tended to increase with time leading to significant differences between the genotypes in older mice in both vulnerable and resilient brain regions. Neither regional ROS damage, measured as HNE-modified protein, nor regional complex I stability, assessed by blue native

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

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Felicitas B. Bidlack

2017-11-01

Full Text Available Mice lacking amelogenin (KO have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle or homozygosity (on both alleles. Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05. The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most

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

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David R Powell

2015-06-01

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

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo.

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Bidlack, Felicitas B; Xia, Yan; Pugach, Megan K

2017-01-01

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes ( Tg ) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180 Tg , CTRNC Tg and LRAP Tg mice to generate M180 Tg and CTRNC Tg double transgene and M180 Tg , CTRNC Tg , LRAP Tg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene ( p structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

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Choi, Soonwook; Yu, Eunah; Lee, Seongwon; Llinás, Rodolfo R.

2015-01-01

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca2+ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states. PMID:26056284

Vaccination of mice for research purpose: alum is as effective as and safer than complete Freund adjuvant

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L. Punzi

2012-12-01

Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disease involving many organ systems. Glomerulonephritis (GLN is one of the major causes of morbidity and mortality in SLE. It has recently been demonstrated that adjuvants of vaccines could cause the so called ASIA syndrome. The study aimed to assess the effects of Complete Freund’s Adjuvant (CFA vs alum injections in NZB/NZWF1 mice. Mice (n=10 each group were injected with a total volume of 200 μL of: CFA in PBS (group 1, alum in PBS (group 2, PBS (group 3 as controls, PTX3/CFA (group 4, PTX3/alum (group 5, 3 times, 3 weeks apart /given in each injection, three weeks apart from ten weeks of age. Urine samples were collected weekly to evaluate proteinuria. Blood samples were collected before every injection, at 21 weeks of age, and at death to evaluate levels of anti-PTX3 and anti-dsDNA. Proteinuria free survival and survival rates were analyzed by the Kaplan-Meier method using Mantel-Cox’s test for comparisons. CFA-treated mice developed both anti-dsDNA antibodies and proteinuria earlier and at higher levels than alumtreated and PBS-injected mice, starting from 13 weeks of age. Proteinuria free survival rates (proteinuria ≥300 mg/dL and survival rates were lower in CFA-treated mice than those treated with alum or injected with PBS (P<0.001 for all. No difference was observed between the alum-treated group and PBS-injected mice. Notably, groups 4 and 5, immunized with PTX3, developed anti-PTX3 antibodies and no significant difference was observed. Alum seems to be as effective as and safer than CFA as adjuvant, since it did not affect disease progression in immunized NZB/NZWF1 mice.

[Combination Chemotherapy Including Intraperitoneal(IP)Administration of Paclitaxel(PTX)followed by PTX, CDDP and S-1Triplet Chemotherapy for CY1P0 Gastric Cancer].

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Shinkai, Masayuki; Imano, Motohiro; Hiraki, Yoko; Kato, Hiroaki; Iwama, Mitsuru; Shiraishi, Osamu; Yasuda, Atsushi; Kimura, Yutaka; Imamoto, Haruhiko; Furukawa, Hiroshi; Yasuda, Takushi

2017-11-01

We evaluate the feasibility and efficacy of combination chemotherapy including single intraperitoneal( IP)administration of paclitaxel(PTX), followed by triplet chemotherapy(PTX, cisplatin[CDDP]and S-1: PCS)for CY1P0 gastric cancer. First of all, we performed staging laparoscopy and confirmed CY1P0, and secondary, administrated PTX intraperitoneally. Thirdly, patients received PCS chemotherapy for 2 courses. After antitumor effect had been confirmed, we performed second look laparoscopy. In the case of CY0P0, we performed gastrectomy with D2 lymph nodes dissection. Total 4 patients were enrolled. Grade 3 leukopenia and neutropenia were observed in one patient while intraperitoneal and systemic-chemotherapy. One patients showed PR and 3 patients showed SD. All patients underwent second look laparoscopy. CY0P0 was observed in all patients and gastrectomy with D2 dissection was performed for all patients. Postoperative complications were observed in 2 patients. Two patients were still alive without recurrence, while the remaining 2 had died of liver metastasis and #16 LN metastasis. Combination chemotherapy including single IP PTX followed by PCS systemic-chemotherapy for CY1P0 gastric cancer is feasible and efficient.

Beta3 adrenoceptors substitute the role of M(2) muscarinic receptor in coping with cold stress in the heart: evidence from M(2)KO mice.

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Benes, Jan; Novakova, Martina; Rotkova, Jana; Farar, Vladimir; Kvetnansky, Richard; Riljak, Vladimir; Myslivecek, Jaromir

2012-07-01

We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

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

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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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

GSK-3α is a central regulator of age-related pathologies in mice.

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Zhou, Jibin; Freeman, Theresa A; Ahmad, Firdos; Shang, Xiying; Mangano, Emily; Gao, Erhe; Farber, John; Wang, Yajing; Ma, Xin-Liang; Woodgett, James; Vagnozzi, Ronald J; Lal, Hind; Force, Thomas

2013-04-01

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

mTOR regulates the expression of DNA damage response enzymes in long-lived Snell dwarf, GHRKO, and PAPPA-KO mice.

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Dominick, Graham; Bowman, Jacqueline; Li, Xinna; Miller, Richard A; Garcia, Gonzalo G

2017-02-01

Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Paclitaxel-Fe3O4 nanoparticles inhibit growth of CD138–  CD34– tumor stem-like cells in multiple myeloma-bearing mice

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Yang C

2013-04-01

Full Text Available Cuiping Yang,1,3,* Jing Wang,2,* Dengyu Chen,1,* Junsong Chen,1 Fei Xiong,4 Hongyi Zhang,1 Yunxia Zhang,2 Ning Gu,4 Jun Dou11Department of Pathogenic Biology and Immunology, Medical School, 2Department of Gynecology and Obstetrics, Zhongda Hospital, Southeast University, Nanjing, 3Department of Pathogenic Biology and Immunology, School of Basic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, 4School of Biological Science and Medical Engineering, Southeast University, Nanjing, People’s Republic of China*These authors contributed equally to this workBackground: There is growing evidence that CD138– CD34– cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138– CD34– tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs on CD138– CD34– tumor stem cells in multiple myeloma-bearing mice.Methods: CD138– CD34– cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated.Results: CD138– CD34– cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 106 CD138– CD34– tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P <  0.005, and the groups that received nanoparticles alone (P < 0.005 or paclitaxel alone (P < 0.05. In addition

Elevated blood pressure in cytochrome P4501A1 knockout mice is associated with reduced vasodilation to omega − 3 polyunsaturated fatty acids

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Agbor, Larry N.; Walsh, Mary T.; Boberg, Jason R.; Walker, Mary K., E-mail: mwalker@salud.unm.edu

2012-11-01

In vitro cytochrome P4501A1 (CYP1A1) metabolizes omega − 3 polyunsaturated fatty acids (n − 3 PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), primarily to 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP), respectively. These metabolites have been shown to mediate vasodilation via increases in nitric oxide (NO) and activation of potassium channels. We hypothesized that genetic deletion of CYP1A1 would reduce vasodilatory responses to n − 3 PUFAs, but not the metabolites, and increase blood pressure (BP) due to decreases in NO. We assessed BP by radiotelemetry in CYP1A1 wildtype (WT) and knockout (KO) mice ± NO synthase (NOS) inhibitor. We also assessed vasodilation to acetylcholine (ACh), EPA, DHA, 17,18-EEQ and 19,20-EDP in aorta and mesenteric arterioles. Further, we assessed vasodilation to an NO donor and to DHA ± inhibitors of potassium channels. CYP1A1 KO mice were hypertensive, compared to WT, (mean BP in mm Hg, WT 103 ± 1, KO 116 ± 1, n = 5/genotype, p < 0.05), and exhibited a reduced heart rate (beats per minute, WT 575 ± 5; KO 530 ± 7; p < 0.05). However, BP responses to NOS inhibition and vasorelaxation responses to ACh and an NO donor were normal in CYP1A1 KO mice, suggesting that NO bioavailability was not reduced. In contrast, CYP1A1 KO mice exhibited significantly attenuated vasorelaxation responses to EPA and DHA in both the aorta and mesenteric arterioles, but normal vasorelaxation responses to the CYP1A1 metabolites, 17,18-EEQ and 19,20-EDP, and normal responses to potassium channel inhibition. Taken together these data suggest that CYP1A1 metabolizes n − 3 PUFAs to vasodilators in vivo and the loss of these vasodilators may lead to increases in BP. -- Highlights: ► CYP1A1 KO mice are hypertensive. ► CYP1A1 KO mice exhibit reduced vasodilation responses to n-3 PUFAs. ► Constitutive CYP1A1 expression regulates blood pressure and vascular function.

Elevated blood pressure in cytochrome P4501A1 knockout mice is associated with reduced vasodilation to omega − 3 polyunsaturated fatty acids

International Nuclear Information System (INIS)

Agbor, Larry N.; Walsh, Mary T.; Boberg, Jason R.; Walker, Mary K.

2012-01-01

In vitro cytochrome P4501A1 (CYP1A1) metabolizes omega − 3 polyunsaturated fatty acids (n − 3 PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), primarily to 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP), respectively. These metabolites have been shown to mediate vasodilation via increases in nitric oxide (NO) and activation of potassium channels. We hypothesized that genetic deletion of CYP1A1 would reduce vasodilatory responses to n − 3 PUFAs, but not the metabolites, and increase blood pressure (BP) due to decreases in NO. We assessed BP by radiotelemetry in CYP1A1 wildtype (WT) and knockout (KO) mice ± NO synthase (NOS) inhibitor. We also assessed vasodilation to acetylcholine (ACh), EPA, DHA, 17,18-EEQ and 19,20-EDP in aorta and mesenteric arterioles. Further, we assessed vasodilation to an NO donor and to DHA ± inhibitors of potassium channels. CYP1A1 KO mice were hypertensive, compared to WT, (mean BP in mm Hg, WT 103 ± 1, KO 116 ± 1, n = 5/genotype, p < 0.05), and exhibited a reduced heart rate (beats per minute, WT 575 ± 5; KO 530 ± 7; p < 0.05). However, BP responses to NOS inhibition and vasorelaxation responses to ACh and an NO donor were normal in CYP1A1 KO mice, suggesting that NO bioavailability was not reduced. In contrast, CYP1A1 KO mice exhibited significantly attenuated vasorelaxation responses to EPA and DHA in both the aorta and mesenteric arterioles, but normal vasorelaxation responses to the CYP1A1 metabolites, 17,18-EEQ and 19,20-EDP, and normal responses to potassium channel inhibition. Taken together these data suggest that CYP1A1 metabolizes n − 3 PUFAs to vasodilators in vivo and the loss of these vasodilators may lead to increases in BP. -- Highlights: ► CYP1A1 KO mice are hypertensive. ► CYP1A1 KO mice exhibit reduced vasodilation responses to n-3 PUFAs. ► Constitutive CYP1A1 expression regulates blood pressure and vascular function.

An Essential Physiological Role for MCT8 in Bone in Male Mice.

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Leitch, Victoria D; Di Cosmo, Caterina; Liao, Xiao-Hui; O'Boy, Sam; Galliford, Thomas M; Evans, Holly; Croucher, Peter I; Boyde, Alan; Dumitrescu, Alexandra; Weiss, Roy E; Refetoff, Samuel; Williams, Graham R; Bassett, J H Duncan

2017-09-01

T3 is an important regulator of skeletal development and adult bone maintenance. Thyroid hormone action requires efficient transport of T4 and T3 into target cells. We hypothesized that monocarboxylate transporter (MCT) 8, encoded by Mct8 on the X-chromosome, is an essential thyroid hormone transporter in bone. To test this hypothesis, we determined the juvenile and adult skeletal phenotypes of male Mct8 knockout mice (Mct8KO) and Mct8D1D2KO compound mutants, which additionally lack the ability to convert the prohormone T4 to the active hormone T3. Prenatal skeletal development was normal in both Mct8KO and Mct8D1D2KO mice, whereas postnatal endochondral ossification and linear growth were delayed in both Mct8KO and Mct8D1D2KO mice. Furthermore, bone mass and mineralization were decreased in adult Mct8KO and Mct8D1D2KO mice, and compound mutants also had reduced bone strength. Delayed bone development and maturation in Mct8KO and Mct8D1D2KO mice is consistent with decreased thyroid hormone action in growth plate chondrocytes despite elevated serum T3 concentrations, whereas low bone mass and osteoporosis reflects increased thyroid hormone action in adult bone due to elevated systemic T3 levels. These studies identify an essential physiological requirement for MCT8 in chondrocytes, and demonstrate a role for additional transporters in other skeletal cells during adult bone maintenance.

Abnormalities in brain structure and behavior in GSK-3alpha mutant mice

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Kaidanovich-Beilin Oksana

2009-11-01

Full Text Available Abstract Background Glycogen synthase kinase-3 (GSK-3 is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. Results Similar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. Conclusion Taken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders.

NLRP3 inflammasome activation mediates fatigue-like behaviors in mice via neuroinflammation.

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Zhang, Ziteng; Ma, Xiujuan; Xia, Zhenna; Chen, Jikuai; Liu, Yangang; Chen, Yongchun; Zhu, Jiangbo; Li, Jinfeng; Yu, Huaiyu; Zong, Ying; Lu, Guocai

2017-09-01

Numerous experimental and clinical studies have suggested that the interaction between the immune system and the brain plays an important role in the pathophysiology of chronic fatigue syndrome (CFS). The NLRP3 inflammasome is an important part of the innate immune system. This complex regulates proinflammatory cytokine interleukin-1β (IL-1β) maturation, which triggers different kinds of immune-inflammatory reactions. We employed repeated forced swims to establish a model of CFS in mice. NLRP3 knockout (KO) mice were also used to explore NLRP3 inflammasome activation in the mechanisms of CFS, using the same treatment. After completing repeated swim tests, the mice displayed fatigue-like behaviors, including locomotor activity and reduced fall-off time on the rota-rod test, which was accompanied by significantly higher mature IL-1β level in the prefrontal cortex (PFC) and malondialdehyde (MDA) level in serum. We also found increased NLRP3 protein expression, NLRP3 inflammasome formation and increased mature IL-1β production in the PFC, relative to untreated mice. The NLRP3 KO mice displayed significantly moderated fatigue behaviors along with decreased PFC and serum IL-1β levels under the same treatment. These findings demonstrated the involvement of NLRP3 inflammasome activation in the mechanism of swimming-induced fatigue. Future therapies targeting the NLRP3/IL-1β pathway may have significant potential for fatigue prevention and treatment. Copyright © 2017. Published by Elsevier Ltd.

Blood-brain barrier disruption in CCL2 transgenic mice during pertussis toxin-induced brain inflammation

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Schellenberg, Angela E; Buist, Richard; Del Bigio, Marc R

2012-01-01

infiltrate into the brain parenchyma following the administration of pertussis toxin (PTx). METHODS: This study uses contrast-enhanced magnetic resonance imaging (MRI) to quantify the extent of blood-brain barrier (BBB) disruption in this model pre- and post-PTx administration compared to wild type mice....... Contrast-enhanced MR images were obtained before and 1, 3, and 5 days after PTx injection in each animal. After the final imaging session fluorescent dextran tracers were administered intravenously to each mouse and brains were examined histologically for cellular infiltrates, BBB leakage and tight...... junction protein. RESULTS: BBB breakdown, defined as a disruption of both the endothelium and glia limitans, was found only in CCL2 transgenic mice following PTx administration seen on MR images as focal areas of contrast enhancement and histologically as dextrans leaking from blood vessels. No evidence...

Reversal of fragile X phenotypes by manipulation of AβPP/Aβ levels in Fmr1KO mice.

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Cara J Westmark

Full Text Available Fragile X syndrome (FXS is the most common form of inherited intellectual disability and the leading known genetic cause of autism. Fragile X mental retardation protein (FMRP, which is absent or expressed at substantially reduced levels in FXS, binds to and controls the postsynaptic translation of amyloid β-protein precursor (AβPP mRNA. Cleavage of AβPP can produce β-amyloid (Aβ, a 39-43 amino acid peptide mis-expressed in Alzheimer's disease (AD and Down syndrome (DS. Aβ is over-expressed in the brain of Fmr1(KO mice, suggesting a pathogenic role in FXS. To determine if genetic reduction of AβPP/Aβ rescues characteristic FXS phenotypes, we assessed audiogenic seizures (AGS, anxiety, the ratio of mature versus immature dendritic spines and metabotropic glutamate receptor (mGluR-mediated long-term depression (LTD in Fmr1(KO mice after removal of one App allele. All of these phenotypes were partially or completely reverted to normal. Plasma Aβ(1-42 was significantly reduced in full-mutation FXS males compared to age-matched controls while cortical and hippocampal levels were somewhat increased, suggesting that Aβ is sequestered in the brain. Evolving therapies directed at reducing Aβ in AD may be applicable to FXS and Aβ may serve as a plasma-based biomarker to facilitate disease diagnosis or assess therapeutic efficacy.

Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice.

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Du, Xianhong; Wu, Zhuanchang; Xu, Yong; Liu, Yuan; Liu, Wen; Wang, Tixiao; Li, Chunyang; Zhang, Cuijuan; Yi, Fan; Gao, Lifen; Liang, Xiaohong; Ma, Chunhong

2018-05-07

As an immune checkpoint, Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases. However, the precise roles of Tim-3 in nonalcoholic steatohepatitis (NASH) remain unstated. In the current study, we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet (MCD)-induced NASH mice. The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice, including F4/80 + CD11b + , F4/80 + CD68 + , and F4/80 + CD169 + macrophages. Remarkably, Tim-3 knockout (KO) significantly accelerated MCD-induced liver steatosis, displaying higher serum ALT, larger hepatic vacuolation, more liver lipid deposition, and more severe liver fibrosis. Moreover, compared with wild-type C57BL/6 mice, Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2, NLRP3, and caspase-1 p20 together with increased generation of IL-1β and IL-18 in livers. In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species (ROS) and related downstream pro-inflammatory cytokine secretion of IL-1β and IL-18 in macrophages. Exogenous administration of N-Acetyl-L-cysteine (NAC), a small molecular inhibitor of ROS, remarkably suppressed caspase-1 p20 expression and IL-1β and IL-18 production in livers of Tim-3 KO mice, thus significantly reducing the severity of steatohepatitis induced by MCD. In conclusion, Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.

The enhanced activity of mass-selected PtxGd nanoparticles for oxygen electroreduction

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Velazquez-Palenzuela, Amado Andres; Masini, Federico; Pedersen, Anders Filsøe

2015-01-01

Mass-selected platinum–gadolinium alloy nanoparticles (PtxGd NPs) are synthesized for the first time as oxygen reduction reaction (ORR) electrocatalysts using the gas aggregation technique, under ultrahigh vacuum (UHV) conditions. The morphology of the PtxGd catalysts is characterized, and their ......Mass-selected platinum–gadolinium alloy nanoparticles (PtxGd NPs) are synthesized for the first time as oxygen reduction reaction (ORR) electrocatalysts using the gas aggregation technique, under ultrahigh vacuum (UHV) conditions. The morphology of the PtxGd catalysts is characterized...

Paradoxical effect of pertussis toxin on the delayed hypersensitivity response to autoantigens in mice.

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Rajwahrdhan Yadav

2010-08-01

Full Text Available Pertussis toxin (PTX, an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU and experimental autoimmune encephalomyelitis (EAE in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens.We have evaluated the effect of PTX on the simultaneous generation of delayed type hypersensitivity (DTH responses and autoimmune responses to uveitogenic interphotoreceptor retinoid binding protein peptide (IRBP161-180, encephalitogenic myelin oligodendrocyte glycoprotein peptide (MOG35-55 or ovalbumin (OVA. PTX injection of mice immunized to IRBP peptide161-180 led to (i the development of EAU as shown by histopathology of the retina, (ii pro-inflammatory cytokine production by splenocytes in response to IRBP peptide161-180, and (iii symptomatic EAE in mice immunized with encephalitogenic MOG peptide35-55. However, mice that received PTX had a reduced DTH response to IRBP161-180 peptide or MOG peptide35-55 when challenged distal to the site affected by autoreactive T cells. Moreover, footpad challenge with MOG35-55 peptide reduced EAE in mice immunized with MOG peptide. In contrast, the use of PTX when immunizing with OVA protein or an OVA immunogenic peptide did not affect the DTH response to OVA.The results suggest that that the reduced DTH response in mice receiving PTX may be specific for autoantigens and autoantigen-reactive T cells are diverted away from ectopic sites that received the autoantigen and towards

The effect of pentoxifylline on L-1 sarcoma tumor growth and angiogenesis in Balb/c mice

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Barbara Joanna Bałan

2017-07-01

Full Text Available Methyloxantines are present in many herbs and vegetal foods, among them in tea, coffee and chocolate. Previous studies revealed that theophylline and theobromine have anti-angiogenic properties. Anti-tumor properties of theobromine were also described. Pentoxifylline (3,7-dimethyl-1-(5-oxohexylxanthine, PTX is a synthetic xanthine derivative. It is a phosphodiesterase inhibitor and has various anti-inflammatory abilities. Pentoxifylline is widely used in therapy of inflammatory arterial diseases such as intermittent claudication of upper and lower limbs as well as in coronary heart disease. The aim of our research was to evaluate the effect of pentoxifylline (individually and in combination with non-steroidal anti-inflammatory drug sulindac, on L-1 sarcoma angiogenic activity and tumor formation in syngeneic Balb/c mice. Pre-incubation of tumor cells for 90 min with various PTX concentrations resulted in dose-dependent decrease of their ability to induce newly-formed blood vessels after transplantation into the skin of recipient mice. Administration of PTX to mice, recipients of tumor cells, slows tumor growth and reduces its volume. Synergistic inhibitory effect of PTX and sulindac, expressed as % of tumors sixth and thirteen day after subcutaneous grafting of L-1 sarcoma into syngeneic Balb/c mice, was observed.

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

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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.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

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Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

2016-01-15

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. Copyright © 2016 the American Physiological Society.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

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Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

2015-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

Fatty acid desaturase 1 knockout mice are lean with improved glycemic control and decreased development of atheromatous plaque

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Powell, David R; Gay, Jason P; Smith, Melinda; Wilganowski, Nathaniel; Harris, Angela; Holland, Autumn; Reyes, Maricela; Kirkham, Laura; Kirkpatrick, Laura L; Zambrowicz, Brian; Hansen, Gwenn; Platt, Kenneth A; van Sligtenhorst, Isaac; Ding, Zhi-Ming; Desai, Urvi

2016-01-01

Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. PMID:27382320

Behavioral Characteristics of Ubiquitin-Specific Peptidase 46-Deficient Mice

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Imai, Saki; Kano, Makoto; Nonoyama, Keiko; Ebihara, Shizufumi

2013-01-01

We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST) and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92), and mice with this mutation (MT mice), as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA) receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT) or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system. PMID:23472206

Behavioral characteristics of ubiquitin-specific peptidase 46-deficient mice.

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Saki Imai

Full Text Available We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92, and mice with this mutation (MT mice, as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system.

Role of pentraxin 3 in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation.

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Suan-Sin Foo

2015-02-01

Full Text Available The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV and Ross River virus (RRV, and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3 was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3(-/- mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis.

Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer

International Nuclear Information System (INIS)

Cao, Qizhen; Li, Zi-Bo; Chen, Kai; Wu, Zhanhong; He, Lina; Chen, Xiaoyuan; Neamati, Nouri

2008-01-01

Targeting drugs to receptors involved in tumor angiogenesis has been demonstrated as a novel and promising approach to improve cancer treatment. In this study, we evaluated the anti-tumor efficacy of a dimeric RGD peptide-paclitaxel conjugate (RGD2-PTX) in an orthotopic MDA-MB-435 breast cancer model. To assess the effect of conjugation and the presence of drug moiety on the MDA-MB-435 tumor and normal tissue uptake, the biodistribution of 3 H-RGD2-PTX was compared with that of 3 H-PTX. The treatment effect of RGD2-PTX and RGD2+PTX was measured by tumor size, 18 F-FDG/PET, 18 F-FLT/PET, and postmortem histopathology. By comparing the biodistribution of 3 H-RGD2-PTX and 3 H-PTX, we found that 3 H-RGD2-PTX had higher initial tumor exposure dose and prolonged tumor retention than 3 H-PTX. Metronomic low-dose treatment of breast cancer indicated that RGD2-PTX is significantly more effective than PTX+RGD2 combination and solvent control. Although in vivo 18 F-FLT/PET imaging and ex vivo Ki67 staining indicated little effect of the PTX-based drug on cell proliferation, 18 F-FDG/PET imaging showed significantly reduced tumor metabolism in the RGD2-PTX-treated mice versus those treated with RGD2+PTX and solvent control. Terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining also showed that RGD2-PTX treatment also had significantly higher cell apoptosis ratio than the other two groups. Moreover, the microvessel density was significantly reduced after RGD2-PTX treatment as determined by CD31 staining. Our results demonstrate that integrin-targeted delivery of paclitaxel allows preferential cytotoxicity to integrin-expressing tumor cells and tumor vasculature. The targeted delivery strategies developed in this study may also be applied to other chemotherapeutics for selective tumor killing. (orig.)

The Growth Hormone Receptor Gene-Disrupted (GHR-KO) Mouse Fails to Respond to an Intermittent Fasting (IF) Diet

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Arum, Oge; Bonkowski, Michael S.; Rocha, Juliana S.; Bartke, Andrzej

2009-01-01

SUMMARY The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long-lived; and this longevity is not responsive to 30% caloric restriction (CR), in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction (DR), we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting (IF) diet. The IF diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction we postulate that GHR-KO mice would be resistant to any manner of DR; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan-extending potential of an intervention. PMID:19747233

In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA

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Pineda-Molina, E.; Daddaoua, A.; Krell, T.; Ramos, J. L.; García-Ruiz, J. M.; Gavira, J. A.

2012-01-01

The crystallization of both native P. putida transcriptional regulator PtxS and its complex with its DNA recognition sequence using the counter-diffusion method are reported. Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space group P3, with unit-cell parameters a = b = 213.71, c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution

Increased anxiety and fear memory in adult mice lacking type 2 deiodinase.

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Bárez-López, Soledad; Montero-Pedrazuela, Ana; Bosch-García, Daniel; Venero, César; Guadaño-Ferraz, Ana

2017-10-01

A euthyroid state in the brain is crucial for its adequate development and function. Impairments in thyroid hormones (THs; T3 or 3,5,3'-triiodothyronine and T4 or thyroxine) levels and availability in brain can lead to neurological alterations and to psychiatric disorders, particularly mood disorders. The thyroid gland synthetizes mainly T4, which is secreted to circulating blood, however, most actions of THs are mediated by T3, the transcriptionally active form. In the brain, intracellular concentrations of T3 are modulated by the activity of type 2 (D2) and type 3 (D3) deiodinases. In the present work, we evaluated learning and memory capabilities and anxiety-like behavior at adult stages in mice lacking D2 (D2KO) and we analyzed the impact of D2-deficiency on TH content and on the expression of T3-dependent genes in the amygdala and the hippocampus. We found that D2KO mice do not present impairments in spatial learning and memory, but they display emotional alterations with increased anxiety-like behavior as well as enhanced auditory-cued fear memory and spontaneous recovery of fear memory following extinction. D2KO mice also presented reduced T3 content in the hippocampus and decreased expression of the T3-dependent gene Dio3 in the amygdala suggesting a hypothyroid status in this structure. We propose that the emotional dysfunctions found in D2KO mice can arise from the reduced T3 content in their brain, which consequently leads to alterations in gene expression with functional consequences. We found a downregulation in the gene encoding for the calcium-binding protein calretinin (Calb2) in the amygdala of D2KO mice that could affect the GABAergic transmission. The current findings in D2KO mice can provide insight into emotional disorders present in humans with DIO2 polymorphisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Abnormal motor phenotype at adult stages in mice lacking type 2 deiodinase.

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Bárez-López, Soledad; Bosch-García, Daniel; Gómez-Andrés, David; Pulido-Valdeolivas, Irene; Montero-Pedrazuela, Ana; Obregon, Maria Jesus; Guadaño-Ferraz, Ana

2014-01-01

Thyroid hormones have a key role in both the developing and adult central nervous system and skeletal muscle. The thyroid gland produces mainly thyroxine (T4) but the intracellular concentrations of 3,5,3'-triiodothyronine (T3; the transcriptionally active hormone) in the central nervous system and skeletal muscle are modulated by the activity of type 2 deiodinase (D2). To date no neurological syndrome has been associated with mutations in the DIO2 gene and previous studies in young and juvenile D2-knockout mice (D2KO) did not find gross neurological alterations, possibly due to compensatory mechanisms. This study aims to analyze the motor phenotype of 3-and-6-month-old D2KO mice to evaluate the role of D2 on the motor system at adult stages in which compensatory mechanisms could have failed. Motor abilities were explored by validated tests. In the footprint test, D2KO showed an altered global gait pattern (mice walked slower, with shorter strides and with a hindlimb wider base of support than wild-type mice). No differences were detected in the balance beam test. However, a reduced latency to fall was found in the rotarod, coat-hanger and four limb hanging wire tests indicating impairment on coordination and prehensile reflex and a reduction of muscle strength. In histological analyses of cerebellum and skeletal muscle, D2KO mice did not present gross structural abnormalities. Thyroid hormones levels and deiodinases activities were also determined. In D2KO mice, despite euthyroid T3 and high T4 plasma levels, T3 levels were significantly reduced in cerebral cortex (48% reduction) and skeletal muscle (33% reduction), but not in the cerebellum where other deiodinase (type 1) is expressed. The motor alterations observed in D2KO mice indicate an important role for D2 in T3 availability to maintain motor function and muscle strength. Our results suggest a possible implication of D2 in motor disorders.

Abnormal motor phenotype at adult stages in mice lacking type 2 deiodinase.

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Soledad Bárez-López

Full Text Available BACKGROUND: Thyroid hormones have a key role in both the developing and adult central nervous system and skeletal muscle. The thyroid gland produces mainly thyroxine (T4 but the intracellular concentrations of 3,5,3'-triiodothyronine (T3; the transcriptionally active hormone in the central nervous system and skeletal muscle are modulated by the activity of type 2 deiodinase (D2. To date no neurological syndrome has been associated with mutations in the DIO2 gene and previous studies in young and juvenile D2-knockout mice (D2KO did not find gross neurological alterations, possibly due to compensatory mechanisms. AIM: This study aims to analyze the motor phenotype of 3-and-6-month-old D2KO mice to evaluate the role of D2 on the motor system at adult stages in which compensatory mechanisms could have failed. RESULTS: Motor abilities were explored by validated tests. In the footprint test, D2KO showed an altered global gait pattern (mice walked slower, with shorter strides and with a hindlimb wider base of support than wild-type mice. No differences were detected in the balance beam test. However, a reduced latency to fall was found in the rotarod, coat-hanger and four limb hanging wire tests indicating impairment on coordination and prehensile reflex and a reduction of muscle strength. In histological analyses of cerebellum and skeletal muscle, D2KO mice did not present gross structural abnormalities. Thyroid hormones levels and deiodinases activities were also determined. In D2KO mice, despite euthyroid T3 and high T4 plasma levels, T3 levels were significantly reduced in cerebral cortex (48% reduction and skeletal muscle (33% reduction, but not in the cerebellum where other deiodinase (type 1 is expressed. CONCLUSIONS: The motor alterations observed in D2KO mice indicate an important role for D2 in T3 availability to maintain motor function and muscle strength. Our results suggest a possible implication of D2 in motor disorders.

Reduced emotional and corticosterone responses to stress in μ-opioid receptor knockout mice

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

2014-01-01

The detailed mechanisms of emotional modulation in the nervous system by opioids remain to be elucidated, although the opioid system is well known to play important roles in the mechanisms of analgesia and drug dependence. In the present study, we conducted behavioral tests of anxiety and depression and measured corticosterone concentrations in both male and female μ-opioid receptor knockout (MOP-KO) mice to reveal the involvement of μ-opioid receptors in stress-induced emotional responses. MOP-KO mice entered more and spent more time in the open arms of the elevated plus maze compared with wild-type mice. MOP-KO mice also displayed significantly decreased immobility in a 15 min tail-suspension test compared with wild-type mice. Similarly, MOP-KO mice exhibited significantly decreased immobility on days 2, 3, and 4 in a 6 min forced swim test conducted for 5 consecutive days. The increase in plasma corticosterone concentration induced by tail-suspension, repeated forced swim, or restraint stress was reduced in MOP-KO mice compared with wild-type mice. Corticosterone levels were not different between wild-type and MOP-KO mice before stress exposure. In contrast, although female mice tended to exhibit fewer anxiety-like responses in the tail-suspension test in both genotypes, no significant gender differences were observed in stress-induced emotional responses. These results suggest that MOPs play an important facilitatory role in emotional responses to stress, including anxiety- and depression-like behavior and corticosterone levels. PMID:19596019

Selective deletion of apolipoprotein E in astrocytes ameliorates the spatial learning and memory deficits in Alzheimer's disease (APP/PS1) mice by inhibiting TGF-β/Smad2/STAT3 signaling.

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Zheng, Jin-Yu; Sun, Jian; Ji, Chun-Mei; Shen, Lin; Chen, Zhong-Jun; Xie, Peng; Sun, Yuan-Zhao; Yu, Ru-Tong

2017-06-01

Astrocytes and apolipoprotein E (apoE) play critical roles in cognitive function, not only under physiological conditions but also in some pathological situations, particularly in the pathological progression of Alzheimer's disease (AD). The regulatory mechanisms underlying the effect of apoE, derived from astrocytes, on cognitive deficits during AD pathology development are unclear. In this study, we generated amyloid precursor protein/apoE knockout (APP/apoE KO ) and APP/glial fibrillary acidic protein (GFAP)-apoE KO mice (the AD mice model used in this study was based on the APP-familial Alzheimer disease overexpression) to investigate the role of apoE, derived from astrocytes, in AD pathology and cognitive function. To explore the mechanism, we investigated the amyloidogenic process related transforming growth factor β/mothers against decapentaplegic homolog 2/signal transducer and activator of transcription 3 (TGF-β/Smad2/STAT3) signaling pathway and further confirmed by administering TGF-β-overexpression adeno-associated virus (specific to astrocytes) to APP/GFAP-apoE KO mice and TGF-β-inhibition adeno-associated virus (specific to astrocytes) to APP/WT mice. Whole body deletion of apoE significantly ameliorated the spatial learning and memory impairment, reduced amyloid β-protein production and inhibited astrogliosis in APP/apoE KO mice, as well as specific deletion apoE in astrocytes in APP/GFAP-apoE KO mice. Moreover, amyloid β-protein accumulation was increased due to promotion of amyloidogenesis of APP, and astrogliosis was upregulated by activation of TGF-β/Smad2/STAT3 signaling. Furthermore, the overexpression of TGF-β in astrocytes in APP/GFAP-apoE KO mice abrogated the effects of apoE knockout. In contrast, repression of TGF-β in astrocytes of APP/WT mice exerted a therapeutic effect similar to apoE knockout. These data suggested that apoE derived from astrocytes contributes to the risk of AD through TGF-β/Smad2/STAT3 signaling activation

Sensorimotor Gating in Neurotensin-1 Receptor Null Mice

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Feifel, D.; Pang, Z.; Shilling, P.D.; Melendez, G.; Schreiber, R.; Button, D.

2009-01-01

BACKGROUND Converging evidence has implicated endogenous neurotensin (NT) in the pathophysiology of brain processes relevant to schizophrenia. Prepulse inhibition of the startle reflex (PPI) is a measure of sensorimotor gating and considered to be of strong relevance to neuropsychiatric disorders associated with psychosis and cognitive dysfunction. Mice genetically engineered to not express NT display deficits in PPI that model the PPI deficits seen in schizophrenia patients. NT1 receptors have been most strongly implicated in mediating the psychosis relevant effects of NT such as attenuating PPI deficits. To investigate the role of NT1 receptors in the regulation of PPI, we measured baseline PPI in wildtype (WT) and NT1 knockout (KO) mice. We also tested the effects of amphetamine and dizocilpine, a dopamine agonist and NMDA antagonist, respectively, that reduce PPI as well as the NT1 selective receptor agonist, PD149163, known to increase PPI in rats. METHODS Baseline PPI and acoustic startle response were measured in WT and NT1 knockout KO mice. After baseline testing, mice were tested again after receiving intraperatoneal (IP) saline or one of three doses of amphetamine (1.0, 3.0 and 10.0 mg/kg), dizocilpine (0.3, 1.0 and 3.0 mg/kg) and PD149163 (0.5, 2.0 and 6.0 mg/kg) on separate test days. RESULTS Baseline PPI and acoustic startle response in NT1 KO mice were not significantly different from NT1 WT mice. WT and KO mice exhibited similar responses to the PPI-disrupting effects of dizocilpine and amphetamine. PD149163 significantly facilitated PPI (P < 0.004) and decreased the acoustic startle response (P < 0.001) in WT but not NT1 KO mice. CONCLUSIONS The data does not support the regulation of baseline PPI or the PPI disruptive effects of amphetamine or dizocilpine by endogenous NT acting at the NT1 receptor, although they support the antipsychotic potential of pharmacological activation of NT1 receptors by NT1 agonists. PMID:19596359

IL-4 deficiency is associated with mechanical hypersensitivity in mice.

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Nurcan Üçeyler

Full Text Available Interleukin-4 (IL-4 is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko mice to characterize their pain behavior before and after chronic constriction injury (CCI of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS of IL-4 ko mice in comparison with wildtype (wt mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001, while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF, IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014. Remarkably, CCI induced TNF (p<0.01, IL-1β (p<0.05, IL-10 (p<0.05, and IL-13 (p<0.001 gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.

Angiotensin-(1-7)/Mas axis modulates fear memory and extinction in mice.

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Lazaroni, Thiago Luiz do Nascimento; Bastos, Cristiane Perácio; Moraes, Márcio Flávio Dutra; Santos, Robson Souza; Pereira, Grace Schenatto

2016-01-01

Inappropriate defense-alerting reaction to fear is a common feature of neuropsychiatric diseases. Therefore, impairments in brain circuits, as well as in molecular pathways underlying the neurovegetative adjustments to fear may play an essential role on developing neuropsychiatric disorders. Here we tested the hypothesis that interfering with angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis homeostasis, which appears to be essential to arterial pressure control, would affect fear memory and extinction. Mas knockout (MasKO) mice, in FVB/N background, showed normal cued fear memory and extinction, but increased freezing in response to context. Next, as FVB/N has poor performance in contextual fear memory, we tested MasKO in mixed 129xC57BL/6 background. MasKO mice behaved similarly to wild-type (WT), but memory extinction was slower in contextual fear conditioning to a weak protocol (1CS/US). In addition, delayed extinction in MasKO mice was even more pronounced after a stronger protocol (3CS/US). We showed previously that Angiotensin II receptor AT1 antagonist, losantan, rescued object recognition memory deficit in MasKO mice. Here, losartan was also effective. Memory extinction was accelerated in MasKO mice after treatment with losartan. In conclusion, we showed for the first time that Ang-(1-7)/Mas axis may modulate fear memory extinction. Furthermore, we suggest MasKO mice as an animal model to study post-traumatic stress disorder (PTSD). Copyright © 2015 Elsevier Inc. All rights reserved.

QDP-JIT/PTX: A QDP++ Implementation for CUDA-Enabled GPUs

Energy Technology Data Exchange (ETDEWEB)

Winter, Frank T. [JLAB; Edwards, Robert G. [JLAB

2014-11-01

These proceedings describe briefly the QDP-JIT/PTX framework for lattice field theory calcula- tions on the CUDA architecture. The framework generates compute kernels in the PTX assembler language which can be compiled to efficient GPU machine code by the NVIDIA JIT compiler. A comprehensive memory management was added to the framework so that applications, e.g. Chroma, can run unaltered on GPU clusters and supercomputers.

Perbandingan Ko-kultur 2D dan 3D dengan Metode Hanging Drop untuk Menghasilkan Micro-environment yang Lebih Relevan Secara Klinis

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Radiana Dhewayani Antarianto

2017-09-01

Full Text Available Ko-kultur sel 2 dimensi (2D kurang menyerupai micro-environment seperti in vivo sedangkan ko-kultur 3 dimensi (3D membentuk mikromassa yang lebih mirip micro-environment in vivo sehingga bermanfaat dalam penelitian biologi dasar. Penelitian ini membandingkan ko-kultur 2D dan 3D sel punca serta sel stelata hepatik dengan metode hanging drop untuk menilai morfologi sel dan pembentukan sferoid dari mikromassa yang terbentuk. Studi in vitro ini dilakukan di Pusat Virologi dan Kanker Patobiologi (PRVKP UI dan laboratorium histologi FKUI pada bulan September 2015 sampai Oktober 2016 menggunakan sel punca yang diisolasi dari darah tali pusat manusia dan sel lestari LX2 (stelata hepatik manusia. Darah tali pusat disortir dengan MACS CD34 dan dianalisis flowcytometry. Ko-kultur sel punca sumsum tulang atau darah tali pusat dan LX2 dilakukan dengan metode hanging drop untuk ko-kultur 2D dan ko-kultur 3D. Triplikasi eksperimen dilakukan untuk tiap set ko-kultur. Hasilnya menunjukkan terdapat perbedaan morfologi ko-kultur 2D dan 3D hanging drop dibandingkan monokultur. Di ko-kultur 2D terdapat mikromassa dan di monokultur 2D tidak terbentuk mikromassa. Di ko-kultur 3D hanging drop, terdapat sferoid yang lebih kecil dibandingkan monokultur 3D hanging drop. Morfologi sel ko-kultur 2D dan 3D dengan metode hanging drop dibandingkan monokultur menunjukkan perubahan fenotip sel-sel yang tergabung dalam mikromassa. Normal 0 false false false false IN X-NONE X-NONE Normal 0 false false false false IN X-NONE X-NONE Fus1 KO Mouse As a Model of Oxidative Stress-Mediated Sporadic Alzheimer's Disease: Circadian Disruption and Long-Term Spatial and Olfactory Memory Impairments.

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Coronas-Samano, Guillermo; Baker, Keeley L; Tan, Winston J T; Ivanova, Alla V; Verhagen, Justus V

2016-01-01

Insufficient advances in the development of effective therapeutic treatments of sporadic Alzheimer's Disease (sAD) to date are largely due to the lack of sAD-relevant animal models. While the vast majority of models do recapitulate AD's hallmarks of plaques and tangles by virtue of tau and/or beta amyloid overexpression, these models do not reflect the fact that in sAD (unlike familial AD) these genes are not risk factors per se and that other mechanisms like oxidative stress, metabolic dysregulation and inflammation play key roles in AD etiology. Here we characterize and propose the Fus1 KO mice that lack a mitochondrial protein Fus1/Tusc2 as a new sAD model. To establish sAD relevance, we assessed sAD related deficits in Fus1 KO and WT adult mice of 4-5 months old, the equivalent human age when the earliest cognitive and olfactory sAD symptoms arise. Fus1 KO mice showed oxidative stress (increased levels of ROS, decreased levels of PRDX1), disruption of metabolic homeostasis (decreased levels of ACC2, increased phosphorylation of AMPK), autophagy (decreased levels of LC3-II), PKC (decreased levels of RACK1) and calcium signaling (decreased levels of Calb2) in the olfactory bulb and/or hippocampus. Mice were behaviorally tested using objective and accurate video tracking (Noldus), in which Fus1 KO mice showed clear deficits in olfactory memory (decreased habituation/cross-habituation in the short and long term), olfactory guided navigation memory (inability to reduce their latency to find the hidden cookie), spatial memory (learning impairments on finding the platform in the Morris water maze) and showed more sleep time during the diurnal cycle. Fus1 KO mice did not show clear deficits in olfactory perception (cross-habituation), association memory (passive avoidance) or in species-typical behavior (nest building) and no increased anxiety (open field, light-dark box) or depression/anhedonia (sucrose preference) at this relatively young age. These neurobehavioral

Odor preference and olfactory memory are impaired in Olfaxin-deficient mice.

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Islam, Saiful; Ueda, Masashi; Nishida, Emika; Wang, Miao-Xing; Osawa, Masatake; Lee, Dongsoo; Itoh, Masanori; Nakagawa, Kiyomi; Tana; Nakagawa, Toshiyuki

2018-06-01

Olfaxin, which is a BNIP2 and Cdc42GAP homology (BCH) domain-containing protein, is predominantly expressed in mitral and tufted (M/T) cells in the olfactory bulb (OB). Olfaxin and Caytaxin, which share 56.3% amino acid identity, are similar in their glutamatergic terminal localization, kidney-type glutaminase (KGA) interaction, and caspase-3 substrate. Although the deletion of Caytaxin protein causes human Cayman ataxia and ataxia in the mutant mouse, the function of Olfaxin is largely unknown. In this study, we generated Prune2 gene mutant mice (Prune2 Ex16-/- ; knock out [KO] mice) using the CRISPR/Cas9 system, during which the exon 16 containing start codon of Olfaxin mRNA was deleted. Exon 16 has 80 nucleotides and is contained in four of five Prune2 isoforms, including PRUNE2, BMCC1, BNIPXL, and Olfaxin/BMCC1s. The levels of Olfaxin mRNA and Olfaxin protein in the OB and piriform cortex of KO mice significantly decreased. Although Prune2 mRNA also significantly decreased in the spinal cord, the gross anatomy of the spinal cord and dorsal root ganglion (DRG) was intact. Further, disturbance of the sensory and motor system was not observed in KO mice. Therefore, in the current study, we examined the role of Olfaxin in the olfactory system where PRUNE2, BMCC1, and BNIPXL are scarcely expressed. Odor preference was impaired in KO mice using opposite-sex urinary scents as well as a non-social odor stimulus (almond). Results of the odor-aversion test demonstrated that odor-associative learning was disrupted in KO mice. Moreover, the NMDAR2A/NMDAR2B subunits switch in the piriform cortex was not observed in KO mice. These results indicated that Olfaxin may play a critical role in odor preference and olfactory memory. Copyright © 2018 Elsevier B.V. All rights reserved.

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism.

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Xing, Lianping; Chen, Di; Boyce, Brendan F

2013-12-01

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.

Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of Fmr1 knockout mice

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Giulia eCuria

2013-04-01

Full Text Available Young, but not adult, Fmr1 knockout (KO mice display audiogenic seizures (AGS that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2 phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT controls at postnatal day (P 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2. Wild running (100% of tested mice followed by clonic/tonic seizures (30% were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P<0.01 vs WT in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P<0.05 vs WT and CA3 (P<0.01. Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P<0.05 vs WT, in both age groups. In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P<0.01 in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.

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

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

2014-01-01

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

Generation and characterization of mice carrying a conditional allele of the Wwox tumor suppressor gene.

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John H Ludes-Meyers

2009-11-01

Full Text Available WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoiesis, leukopenia, and splenic atrophy. Impaired hematopoiesis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues.

Food-induced reinforcement is abrogated by the genetic deletion of the MT1 or MT2 melatonin receptor in C3H/HeN mice.

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Clough, Shannon J; Hudson, Randall L; Dubocovich, Margarita L

2018-05-02

Palatable food is known for its ability to enhance reinforcing responses. Studies have suggested a circadian variation in both drug and natural reinforcement, with each following its own time course. The goal of this study was to determine the role of the MT 1 and MT 2 melatonin receptors in palatable snack food-induced reinforcement, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for snack food-induced CPP at either ZT 6 - 8 (ZT: Zeitgeber time; ZT 0 = lights on), when endogenous melatonin levels are low, or ZT 19 - 21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The amount of snack food (chow, Cheetos®, Froot Loops® and Oreos®) consumed was of similar magnitude at both times, however only C3H/HeN mice conditioned to snack food at ZT 6 - 8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT 1 (MT 1 KO) or MT 2 (MT 2 KO) receptor tested at ZT 6 - 8 did not develop a place preference for snack food. Although the MT 2 KO mice showed a similar amount of snack food consumed when compared to wild-type mice, the MT 1 KO mice consumed significantly less than either genotype. We conclude that in our mouse model snack food-induced CPP is dependent on time of day and the presence of the MT 1 or MT 2 receptors, suggesting a role for melatonin and its receptors in snack food-induced reinforcement. Copyright © 2018 Elsevier B.V. All rights reserved.

Fus1 KO mouse as a model of oxidative stress-mediated sporadic Alzheimer’s disease: circadian disruption and long-term spatial and olfactory memory impairments.

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Guillermo Coronas-Samano

2016-11-01

Full Text Available Insufficient advances in the development of effective therapeutic treatments of sporadic Alzheimer's Disease (sAD to date are largely due to the lack of sAD-relevant animal models. While the vast majority of models do recapitulate AD's hallmarks of plaques and tangles by virtue of tau and/or beta amyloid overexpression, these models do not reflect the fact that in sAD (unlike familial AD these genes are not risk factors per se and that other mechanisms like oxidative stress, metabolic dysregulation and inflammation play key roles in AD etiology. Here we characterize and propose the Fus1 KO mice that lack a mitochondrial protein Fus1/Tusc2 as a new sAD model. To establish sAD relevance, we assessed sAD related deficits in Fus1 KO and WT adult mice of 4-5 months old, the equivalent human age when the earliest cognitive and olfactory sAD symptoms arise. Fus1 KO mice showed oxidative stress (increased levels of ROS, decreased levels of PRDX1, disruption of metabolic homeostasis (decreased levels of ACC2, increased phosphorylation of AMPK, autophagy (decreased levels of LC3-II, PKC (decreased levels of RACK1 and calcium signaling (decreased levels of Calb2 in the olfactory bulb and/or hippocampus. Mice were behaviorally tested using objective and accurate video tracking (Noldus, in which Fus1 KO mice showed clear deficits in olfactory memory (decreased habituation/cross-habituation in the short and long term, olfactory guided navigation memory (inability to reduce their latency to find the hidden cookie, spatial memory (learning impairments on finding the platform in the Morris water maze and showed more sleep time during the diurnal cycle. Fus1 KO mice did not show clear deficits in olfactory perception (cross-habituation, association memory (passive avoidance or in species-typical behavior (nest building and no increased anxiety (open field, light-dark box or depression/anhedonia (sucrose preference at this relatively young age. These

Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice.

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Fan, Fan; Cao, Quan; Wang, Cong; Ma, Xin; Shen, Cheng; Liu, Xiang-wei; Bu, Li-ping; Zou, Yun-zeng; Hu, Kai; Sun, Ai-jun; Ge, Jun-bo

2014-08-01

To investigate the roles of acetaldehyde dehydrogenase 2 (ALDH2), the key enzyme of ethanol metabolism, in chronic low to moderate alcohol consumption-induced heart protective effects in mice. Twenty-one male wild-type (WT) or ALDH2-knockout (KO) mice were used in this study. In each genotype, 14 animals received alcohol (2.5%, 5% and 10% in week 1-3, respectively, and 18% in week 4-7), and 7 received water for 7 weeks. After the treatments, survival rate and general characteristics of the animals were evaluated. Serum ethanol and acetaldehyde levels and blood lipids were measured. Metabolomics was used to characterize the heart and serum metabolism profiles. Chronic alcohol intake decreased the survival rate of KO mice by 50%, and significantly decreased their body weight, but did not affect those of WT mice. Chronic alcohol intake significantly increased the serum ethanol levels in both WT and KO mice, but KO mice had significantly higher serum acetaldehyde levels than WT mice. Chronic alcohol intake significantly increased the serum HDL cholesterol levels in WT mice, and did not change the serum HDL cholesterol levels in KO mice. After chronic alcohol intake, WT and KO mice showed differential heart and serum metabolism profiles, including the 3 main energy substrate types (lipids, glucose and amino acids) and three carboxylic acid cycles. Low to moderate alcohol consumption increases HDL cholesterol levels and improves heart energy metabolism profile in WT mice but not in ALDH2-KO mice. Thus, preserved ALDH2 function is essential for the protective effect of low to moderate alcohol on the cardiovascular system.

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity.

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Alessandro Marsili

Full Text Available The type 2 iodothyronine deiodinase (D2 converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT, and mice with a disrupted Dio2 gene (D2KO have an impaired response to cold. BAT is also activated by overfeeding.After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V0(2 was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER, suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance.We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.

Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration

International Nuclear Information System (INIS)

Chowdhury, Uttam K.; Zakharyan, Robert A.; Hernandez, Alba; Avram, Mihaela D.; Kopplin, Michael J.; Aposhian, H. Vasken

2006-01-01

Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic + 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested. The livers of male knockout (KO) mice, in which 222 bp of Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate

The Ghrelin/GOAT System Regulates Obesity-Induced Inflammation in Male Mice.

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Harvey, Rebecca E; Howard, Victor G; Lemus, Moyra B; Jois, Tara; Andrews, Zane B; Sleeman, Mark W

2017-07-01

Ghrelin plays a key role in appetite, energy homeostasis, and glucose regulation. Recent evidence suggests ghrelin suppresses inflammation in obesity; however, whether this is modulated by the acylated and/or des-acylated peptide is unclear. We used mice deficient in acylated ghrelin [ghrelin octanoyl-acyltransferase (GOAT) knockout (KO) mice], wild-type (WT) littermates, and C57BL/6 mice to examine the endogenous and exogenous effects of acyl and des-acyl ghrelin on inflammatory profiles under nonobese and obese conditions. We demonstrate that in the spleen, both ghrelin and GOAT are localized primarily in the red pulp. Importantly, in the thymus, ghrelin was predominantly localized to the medulla, whereas GOAT was found in the cortex, implying differing roles in T cell development. Acute exogenous treatment with acyl/des-acyl ghrelin suppressed macrophage numbers in spleen and thymus in obese mice, whereas only acyl ghrelin increased CD3+ T cells in the thymus in mice fed both chow and a high-fat-diet (HFD). Consistent with this result, macrophages were increased in the spleen of KO mice on a HFD. Whereas there was no difference in CD3+ T cells in the plasma, spleen, or thymus of WT vs KO mice, KO chow and HFD-fed mice displayed decreased leukocytes. Our results suggest that the acylation status affects the anti-inflammatory properties of ghrelin under chow and HFD conditions. Copyright © 2017 Endocrine Society.

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

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

2018-01-15

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.

Differential response of nNOS knockout mice to MDMA ("ecstasy")- and methamphetamine-induced psychomotor sensitization and neurotoxicity.

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Itzhak, Yossef; Anderson, Karen L; Ali, Syed F

2004-10-01

It has been shown that mice deficient in neuronal nitric oxide synthase (nNOS) gene are resistant to cocaine-induced psychomotor sensitization and methamphetamine (METH)-induced dopaminergic neurotoxicity. The present study was undertaken to investigate the hypothesis that nNOS has a major role in dopamine (DA)- but not serotonin (5-hydroxytryptamine; 5-HT)-mediated effects of psychostimulants. The response of nNOS knockout (KO) and wild-type (WT) mice to the psychomotor-stimulating and neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") and METH were investigated. Repeated administration of MDMA for 5 days resulted in psychomotor sensitization in both WT and nNOS KO mice, while repeated administration of METH caused psychomotor sensitization in WT but not in KO mice. Sensitization to both MDMA and METH was persistent for 40 days in WT mice, but not in nNOS KO mice. These findings suggest that the induction of psychomotor sensitization to MDMA and METH is NO independent and NO dependent, respectively, while the persistence of sensitization to both drugs is NO dependent. For the neurochemical studies, a high dose of MDMA caused marked depletion of 5-HT in several brain regions of both WT and KO mice, suggesting that the absence of the nNOS gene did not afford protection against MDMA-induced depletion of 5-HT. Striatal dopaminergic neurotoxicity caused by high doses of MDMA and METH in WT mice was partially prevented in KO mice administered with MDMA, but it was fully precluded in KO mice administered with METH. The differential response of nNOS KO mice to the behavioral and neurotoxic effects of MDMA and METH suggests that the nNOS gene is required for the expression and persistence of DA-mediated effects of METH and MDMA, while 5-HT-mediated effects of MDMA (induction of sensitization and 5-HT depletion) are not dependent on nNOS.

Experimental Demyelination and Axonal Loss Are Reduced in MicroRNA-146a Deficient Mice.

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Martin, Nellie A; Molnar, Viktor; Szilagyi, Gabor T; Elkjaer, Maria L; Nawrocki, Arkadiusz; Okarmus, Justyna; Wlodarczyk, Agnieszka; Thygesen, Eva K; Palkovits, Miklos; Gallyas, Ferenc; Larsen, Martin R; Lassmann, Hans; Benedikz, Eirikur; Owens, Trevor; Svenningsen, Asa F; Illes, Zsolt

2018-01-01

The cuprizone (CPZ) model of multiple sclerosis (MS) was used to identify microRNAs (miRNAs) related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO) mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs) during remyelination, but its role has not been examined during demyelination. MicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray) and proteome (liquid chromatography tandem mass spectrometry) of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis. miR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP + oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2 + OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3 + and Iba1 + macrophages/microglia was

Host resistance of CD18 knockout mice against systemic infection with Listeria monocytogenes

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Wu, Huaizhu; Prince, Joseph E.; Brayton, Cory F.; Shah, Chirayu; Zeve, Daniel; Gregory, Stephen H.; Smith, C. Wayne; Ballantyne, Christie M.

2003-01-01

Mice with targeted mutations of CD18, the common beta2 subunit of CD11/CD18 integrins, have leukocytosis, impaired transendothelial neutrophil emigration, and reduced host defense to Streptococcus pneumoniae, a gram-positive extracellular bacterium. Previous studies using blocking monoclonal antibodies suggested roles for CD18 and CD11b in hepatic neutrophil recruitment and host innate response to Listeria monocytogenes, a gram-positive intracellular bacterium. We induced systemic listeriosis in CD18 knockout (CD18-ko) and wild-type (WT) mice by tail vein injection with Listeria. By 14 days postinjection (dpi), 8 of 10 WT mice died, compared with 2 of 10 CD18-ko mice (P Listeria organisms in livers and spleens were similar in both groups at 20 min postinfection. By 3, 5, and 7 dpi, however, numbers of Listeria organisms were significantly lower in livers and spleens of CD18-ko mice than in WT mice. Histopathology showed that following Listeria infection, CD18-ko mice had milder inflammatory and necrotizing lesions in both spleens and livers than did WT mice. Cytokine assays indicated that baseline interleukin-1beta and granulocyte colony-stimulating factor (G-CSF) levels were higher in CD18-ko mice than in WT mice and that CD18-ko splenocytes produced higher levels of interleukin-1beta and G-CSF than WT splenocytes under the same amount of Listeria stimulation. These findings show that CD18 is not an absolute requirement for antilisterial innate immunity or hepatic neutrophil recruitment. We propose that the absence of CD18 in the mice results in the priming of innate immunity, as evidenced by elevated cytokine expression, and neutrophilic leukocytosis, which augments antilisterial defense.

Permanent electric dipole moments of PtX (X = H, F, Cl, Br, and I) by the composite approach

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Deng, Dan; Lian, Yongqin; Zou, Wenli

2017-11-01

Using the FPD composite approach of Peterson et. al. we calculate the permanent electric dipole moments of PtX (X = H, F, Cl, Br, and I) at the equilibrium geometries of their ground states. The dipole moment of PtF is estimated to be 3.421 Debye, being very close to the experimental value of 3.42(6) Debye. This research also suggests the ordering of dipole moments of PtX being proportional to the electronegativity of X.

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication.

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Dunn, Emily N; Ferrara-Bowens, Teresa M; Chachich, Mark E; Honnold, Cary L; Rothwell, Cristin C; Hoard-Fruchey, Heidi M; Lesyna, Catherine A; Johnson, Erik A; Cerasoli, Douglas M; McDonough, John H; Cadieux, C Linn

2018-06-07

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Seipin knockout in mice impairs stem cell proliferation and progenitor cell differentiation in the adult hippocampal dentate gyrus via reduced levels of PPARγ

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Guoxi Li

2015-12-01

Full Text Available The seipin gene (BSCL2 was originally identified in humans as a loss-of-function gene associated with congenital generalized lipodystrophy type 2 (CGL2. Neuronal seipin-knockout (seipin-nKO mice display a depression-like phenotype with a reduced level of hippocampal peroxisome proliferator-activated receptor gamma (PPARγ. The present study investigated the influence of seipin deficiency on adult neurogenesis in the hippocampal dentate gyrus (DG and the underlying mechanisms of the effects. We show that the proliferative capability of stem cells in seipin-nKO mice was substantially reduced compared to in wild-type (WT mice, and that this could be rescued by the PPARγ agonist rosiglitazone (rosi. In seipin-nKO mice, neuronal differentiation of progenitor cells was inhibited, with the enhancement of astrogliogenesis; both of these effects were recovered by rosi treatment during early stages of progenitor cell differentiation. In addition, rosi treatment could correct the decline in hippocampal ERK2 phosphorylation and cyclin A mRNA level in seipin-nKO mice. The MEK inhibitor U0126 abolished the rosi-rescued cell proliferation and cyclin A expression in seipin-nKO mice. In seipin-nKO mice, the hippocampal Wnt3 protein level was less than that in WT mice, and there was a reduction of neurogenin 1 (Neurog1 and neurogenic differentiation 1 (NeuroD1 mRNA, levels of which were corrected by rosi treatment. STAT3 phosphorylation (Tyr705 was enhanced in seipin-nKO mice, and was further elevated by rosi treatment. Finally, rosi treatment for 10 days could alleviate the depression-like phenotype in seipin-nKO mice, and this alleviation was blocked by the MEK inhibitor U0126. The results indicate that, by reducing PPARγ, seipin deficiency impairs proliferation and differentiation of neural stem and progenitor cells, respectively, in the adult DG, which might be responsible for the production of the depression-like phenotype in seipin-nKO mice.

Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.

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Lambert, G; Sakai, N; Vaisman, B L; Neufeld, E B; Marteyn, B; Chan, C C; Paigen, B; Lupia, E; Thomas, A; Striker, L J; Blanchette-Mackie, J; Csako, G; Brady, J N; Costello, R; Striker, G E; Remaley, A T; Brewer, H B; Santamarina-Fojo, S

2001-05-04

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Paclitaxel-induced hypothermia and hypoperfusion increase breast cancer metastasis and angiogenesis in mice

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Ami, Nozomi; Sato, Hideki; Hayakawa, Yoshihiro

2018-01-01

Housing temperature has been shown to influence thermoregulation and behavior of preclinical cancer models; and anti-cancer drugs typically reduce peripheral blood flow and body temperature. In the present study, the effects of paclitaxel (PTX)-induced reduction of body temperature and peripheral blood flow on metastatic 4T1 breast cancer was investigated in a mouse model and the modification of these effects by thermoneutral temperature was also assessed. A single dose of PTX decreased the body temperature and peripheral blood flow in mice housed at a standard temperature (23°C). Furthermore, although lung metastasis and angiogenesis of inoculated 4T1 cells increased in mice pretreated with PTX, mice housed at a thermoneutral temperature (30°C) could compensate their body temperature and peripheral blood flow compared with control mice, and also suppressed 4T1 angiogenesis and metastasis to lung. The present results imply that maintenance of body temperature or efficient energy supply for thermogenesis may prevent tumor relapse or metastasis after chemotherapy. PMID:29434941

Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice

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Mingam, Rozenn; Moranis, Aurélie; Bluthé, Rose-Marie; De Smedt-Peyrusse, Véronique; Kelley, Keith W.; Guesnet, Philippe; Lavialle, Monique; Dantzer, Robert; Layé, Sophie

2009-01-01

Sickness behaviour is an adaptive behavioural response to the activation of the innate immune system. It is mediated by brain cytokine production and action, especially interleukin-6 (IL-6). Polyunsaturated fatty acids (PUFA) are essential fatty acids that are highly incorporated in brain cells membranes and display immunomodulating properties. We hypothesized that a decrease in n-3 PUFA brain level by dietary means impacts on lipopolysaccharide (LPS)-induced IL-6 production and sickness behaviour. Our results show that mice exposed throughout life to a diet containing n-3 PUFA (n-3/n-6 diet) display a decrease in social interaction that does not occur in mice submitted to a diet devoid of n-3 PUFA (n-6 diet). LPS induced high IL-6 plasma levels as well as expression of IL-6 mRNA in the hippocampus and cFos mRNA in the brainstem of mice fed either diet, indicating intact immune-to-brain communication. However, STAT3 and STAT1 activation, a hallmark of IL-6 signalling pathway, was lower in the hippocampus of LPS-treated n-6 mice as compared to n-3/n-6 mice. In addition, LPS did not reduce social interaction in IL-6 knock-out (IL-6 KO) mice and failed to induce STAT3 activation in the brain of IL-6 KO mice. Altogether, these findings point to alteration in brain STAT3 as a key mechanism for the lack of effect of LPS on social interaction in mice fed with the n-6 PUFA diet. The relative deficiency of Western diets in n-3 PUFA could impact on behavioural aspects of the host response to infection. PMID:18973601

PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

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Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

2009-01-01

The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO......) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1alpha KO mice, VEGF protein...... expression was approximately 60-80% lower and the capillary-to-fiber ratio approximately 20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1alpha KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased...

Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance.

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She, Pengxiang; Zhou, Yingsheng; Zhang, Zhiyou; Griffin, Kathleen; Gowda, Kavitha; Lynch, Christopher J

2010-04-01

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43-79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and alpha-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Experimental Demyelination and Axonal Loss Are Reduced in MicroRNA-146a Deficient Mice

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Nellie A. Martin

2018-03-01

Full Text Available BackgroundThe cuprizone (CPZ model of multiple sclerosis (MS was used to identify microRNAs (miRNAs related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs during remyelination, but its role has not been examined during demyelination.MethodsMicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray and proteome (liquid chromatography tandem mass spectrometry of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis.ResultsmiR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP+ oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2+ OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3+ and

Okoljski vplivi planinskih koč v visokogorju Kamniško-Savinjskih Alp

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Nejc Bobovnik

2014-12-01

Full Text Available Planinske koče so v neposeljenem gorskem svetu skoraj edini lokalni vir onesnaževanja. V raziskavi smo opredelili štiri okoljske vplive planinskih koč: oskrbovanje in ravnanje z odpadki, ogrevanje in pridobivanje električne energije, oskrba z vodo in ravnanje z odpadnimi vodami ter število in obnašanje obiskovalcev. S pomočjo ankete smo ocenili okoljske vplive izbranih planinskih koč. Najbolj problematična sta velika količina odpadkov in porabljene vode, kot dobro lahko označimo oskrbovanje, kot zelo dobro ogrevanje koč in pridobivanje električne energije.

Role of surfactant protein-A (SP-A) in lung injury in response to acute ozone exposure of SP-A deficient mice

International Nuclear Information System (INIS)

Haque, Rizwanul; Umstead, Todd M.; Ponnuru, Padmavathi; Guo Xiaoxuan; Hawgood, Samuel; Phelps, David S.; Floros, Joanna

2007-01-01

Millions are exposed to ozone levels above recommended limits, impairing lung function, causing epithelial damage and inflammation, and predisposing some individuals to pneumonia, asthma, and other lung conditions. Surfactant protein-A (SP-A) plays a role in host defense, the regulation of inflammation, and repair of tissue damage. We tested the hypothesis that the lungs of SP-A(-/-) (KO) mice are more susceptible to ozone-induced damage. We compared the effects of ozone on KO and wild type (WT) mice on the C57BL/6 genetic background by exposing them to 2 parts/million of ozone for 3 or 6 h and sacrificing them 0, 4, and 24 h later. Lungs were subject to bronchoalveolar lavage (BAL) or used to measure endpoints of oxidative stress and inflammation. Despite more total protein in BAL of KO mice after a 3 h ozone exposure, WT mice had increased oxidation of protein and had oxidized SP-A dimers. In KO mice there was epithelial damage as assessed by increased LDH activity and there was increased phospholipid content. In WT mice there were more BAL PMNs and elevated macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1. Changes in MIP-2 and MCP-1 were observed in both KO and WT, however mRNA levels differed. In KO mice MIP-2 mRNA levels changed little with ozone, but in WT levels they were significantly increased. In summary, several aspects of the inflammatory response differ between WT and KO mice. These in vivo findings appear to implicate SP-A in regulating inflammation and limiting epithelial damage in response to ozone exposure

Increased anxiety-related behaviour in Hint1 knockout mice.

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Varadarajulu, Jeeva; Lebar, Maria; Krishnamoorthy, Gurumoorthy; Habelt, Sonja; Lu, Jia; Bernard Weinstein, I; Li, Haiyang; Holsboer, Florian; Turck, Christoph W; Touma, Chadi

2011-07-07

Several reports have implicated a role for the histidine triad nucleotide-binding protein-1 (Hint1) in psychiatric disorders. We have studied the emotional behaviour of male Hint1 knockout (Hint1 KO) mice in a battery of tests and performed biochemical analyses on brain tissue. The behavioural analysis revealed that Hint1 KO mice exhibit an increased emotionality phenotype compared to wildtype (WT) mice, while no significant differences in locomotion or general exploratory activity were noted. In the elevated plus-maze (EPM) test, the Hint1 KO animals entered the open arms of the apparatus less often than WT littermates. Similarly, in the dark-light box test, Hint1 KO mice spent less time in the lit compartment and the number of entries were reduced, which further confirmed an increased anxiety-related behaviour. Moreover, the Hint1 KO animals showed significantly more struggling and less floating behaviour in the forced swim test (FST), indicating an increased emotional arousal in aversive situations. Hint1 is known as a protein kinase C (PKC) interacting protein. Western blot analysis showed that PKCγ expression was elevated in Hint1 KO compared to WT mice. Interestingly, PKCγ mRNA levels of the two groups did not show a significant difference, implying a post-transcriptional PKCγ regulation. In addition, PKC enzymatic activity was increased in Hint1 KO compared to WT mice. In summary, our results indicate a role for Hint1 and PKCγ in modulating anxiety-related and stress-coping behaviour in mice. Copyright © 2011 Elsevier B.V. All rights reserved.

Diacylglycerol kinase β knockout mice exhibit attention-deficit behavior and an abnormal response on methylphenidate-induced hyperactivity.

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Mitsue Ishisaka

Full Text Available BACKGROUND: Diacylglycerol kinase (DGK is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density, hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD. METHODOLOGY/PRINCIPAL FINDINGS: DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.. In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p., but showed a similar response to an N-methyl-d-aspartate (NMDA receptor antagonist, MK-801 (0.3 mg/kg, i.p., when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK, which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum.

Long-term activation of TLR3 by Poly(I:C induces inflammation and impairs lung function in mice

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Alexopoulou Lena

2009-06-01

Full Text Available Abstract Background The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. Methods TLR3 knock-out (KO mice and C57B6 (WT mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C. Results There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFα were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C, the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. Conclusion These findings demonstrate that TLR3 activation by poly(I:C modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

Intravenous administration of high-dose Paclitaxel reduces gut-associated lymphoid tissue cell number and respiratory immunoglobulin A concentrations in mice.

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Moriya, Tomoyuki; Fukatsu, Kazuhiko; Noguchi, Midori; Okamoto, Koichi; Murakoshi, Satoshi; Saitoh, Daizoh; Miyazaki, Masaru; Hase, Kazuo; Yamamoto, Junji

2014-02-01

Chemotherapy remains a mainstay of treatment for cancer patients. However, anti-cancer drugs frequently cause a wide range of side effects, including leukopenia and gastrointestinal toxicity. These adverse effects can lead to treatment delays or necessitate temporary dose reductions. Although chemotherapy-related changes in gut morphology have been demonstrated, the influences of chemotherapeutic regimens on gut immunity are understood poorly. This study aimed to examine whether the anti-cancer drug paclitaxel (PTX) impairs gut immunity in mice. Male ICR mice were randomized into three groups: Control, low-dose PTX (low PTX; 2 mg/kg), or high-dose PTX (high PTX; 4 mg/kg). A single intravenous dose was given. On day seven after the injection, lymphocytes from Peyer patches (PP), intraepithelial (IE) spaces, and the lamina propria (LP) were counted and analyzed by flow cytometry (CD4(+), CD8(+), αβTCR(+), γδTCR(+), B220(+)). Immunoglobulin A (IgA) concentrations were measured in small intestinal and respiratory tract washings. Total, CD4(+) and γδTCR(+) lymphocyte numbers in PPs were significantly lower in the high PTX than in the control group. The CD4(+) lymphocyte numbers in the IE spaces were significantly lower in both PTX groups than in the control group. Respiratory tract IgA concentrations were lower in the high PTX than in the control group. The present data suggest high-dose PTX impairs mucosal immunity, possibly rendering patients more vulnerable to infection. Careful dose selection and new therapies may be important for maintaining mucosal immunity during PTX chemotherapy.

Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition

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Zhu Zhu

2016-01-01

Full Text Available Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice by altering exposure to light. C57 BL/6J mice (C57 mice and ApoE-KO mice (ApoE-KO mice exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1 levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation.

Relationship between Sustained Reductions in Plasma Lipid and Lipoprotein Concentrations with Apheresis and Plasma Levels and mRNA Expression of PTX3 and Plasma Levels of hsCRP in Patients with HyperLp(alipoproteinemia

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Claudia Stefanutti

2016-01-01

Full Text Available The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI (LA on plasma levels of pentraxin 3 (PTX3, an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(alipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged 55±9.3 years (mean ± SD, were enrolled in the study. PTX3 soluble protein levels in plasma were unchanged by 2 sessions of LA; however, a downregulation of mRNA expression for PTX3 was observed, starting with the first session of LA (p<0.001. The observed reduction was progressively increased in the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session. A statistically significantly greater treatment-effect correlation was observed in patients undergoing weekly treatments, compared with those undergoing treatment every 15 days. A progressive reduction in plasma levels of C-reactive protein was also seen from the first session of LA, with a statistically significant linear correlation for treatment-effect in the change in plasma levels of this established inflammatory marker (R2=0.99; p<0.001. Our findings suggest that LA has anti-inflammatory and endothelium protective effects beyond its well-established efficacy in lowering apoB100-containing lipoproteins.

Rearing-environment-dependent hippocampal local field potential differences in wild-type and inositol trisphosphate receptor type 2 knockout mice.

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Tanaka, Mika; Wang, Xiaowen; Mikoshiba, Katsuhiko; Hirase, Hajime; Shinohara, Yoshiaki

2017-10-15

Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca 2+ surges are involved in this experience-dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP 3 R2)-knockout (KO) mice, in which IP 3 /Ca 2+ signalling in astrocytes is largely diminished. We found that this experience-dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP 3 R2 deficiency. Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression-like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane-anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non-theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca 2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP 3 R2)-knockout (KO) mice, in which astrocytic Ca 2+ elevations are largely diminished. We found that the gamma power was also higher in IP 3 R2-KO-ENR mice compared to IP 3 R2-KO-ISO mice, suggesting that the rearing-environment-dependent gamma power alteration does not necessarily

Human Albumin Fragments Nanoparticles as PTX Carrier for Improved Anti-cancer Efficacy

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Liang Ge

2018-06-01

Full Text Available For enhanced anti-cancer performance, human serum albumin fragments (HSAFs nanoparticles (NPs were developed as paclitaxel (PTX carrier in this paper. Human albumins were broken into fragments via degradation and crosslinked by genipin to form HSAF NPs for better biocompatibility, improved PTX drug loading and sustained drug release. Compared with crosslinked human serum albumin NPs, the HSAF-NPs showed relative smaller particle size, higher drug loading, and improved sustained release. Cellular and animal results both indicated that the PTX encapsulated HSAF-NPs have shown good anti-cancer performance. And the anticancer results confirmed that NPs with fast cellular internalization showed better tumor inhibition. These findings will not only provide a safe and robust drug delivery NP platform for cancer therapy, but also offer fundamental information for the optimal design of albumin based NPs.

Sarcocystis jamaicensis n. sp., from Red-Tailed Hawks (Buteo jamaicensis) Definitive Host and IFN-γ Gene Knockout Mice as Experimental Intermediate Host.

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Verma, S K; von Dohlen, A Rosypal; Mowery, J D; Scott, D; Rosenthal, B M; Dubey, J P; Lindsay, D S

2017-10-01

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-γ gene knockout (KO) mice as an experimental intermediate host in which sarcocysts form in muscle. Two RTHs submitted to the Carolina Raptor Center, Huntersville, North Carolina, were euthanized because they could not be rehabilitated and released. Fully sporulated 12.5 × 9.9-μm sized sporocysts were found in intestinal scrapings of both hawks. Sporocysts were orally fed to laboratory-reared outbred Swiss Webster mice (SW, Mus musculus) and also to KO mice. The sporocysts were infective for KO mice but not for SW mice. All SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in any SW mice euthanized on days 54, 77, 103 (n = 2) or 137 post-inoculation (PI). The KO mice developed neurological signs and were necropsied between 52 to 68 days PI. Schizonts/merozoites were found in all KO mice euthanized on days 52, 55 (n = 3), 59, 61 (n = 2), 66, and 68 PI and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neural tissue. The schizonts/merozoites were located in neural tissue and were apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice by subcutaneous inoculation and when seeded on to CV-1 cells. Microscopic sarcocysts were found in skeletal muscles of 5 of 8 KO mice euthanized between 55-61 days PI. Only a few sarcocysts were detected. Sarcocysts were microscopic, up to 3.5 mm long. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 μm thick) and smooth. By transmission electron microscopy, the sarcocyst wall classified as "type 1j" (new designation). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes revealed a close relationship with Sarcocystis microti and Sarcocystis glareoli; both species infect birds as definitive hosts

Toll-like receptor 2 promotes neurogenesis from the dentate gyrus after photothrombotic cerebral ischemia in mice.

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Seong, Kyung-Joo; Kim, Hyeong-Jun; Cai, Bangrong; Kook, Min-Suk; Jung, Ji-Yeon; Kim, Won-Jae

2018-03-01

The subgranular zone (SGZ) of hippocampal dentate gyrus (HDG) is a primary site of adult neurogenesis. Toll-like receptors (TLRs), are involved in neural system development of Drosophila and innate immune response of mammals. TLR2 is expressed abundantly in neurogenic niches such as adult mammalian hippocampus. It regulates adult hippocampal neurogenesis. However, the role of TLR2 in adult neurogenesis is not well studied in global or focal cerebral ischemia. Therefore, this study aimed to investigate the role of TLR2 in adult neurogenesis after photochemically induced cerebral ischemia. At 7 days after photothrombotic ischemic injury, the number of bromodeoxyuridine (BrdU)-positive cells was increased in both TLR2 knock-out (KO) mice and wild-type (WT) mice. However, the increment rate of BrdU-positive cells was lower in TLR2 KO mice compared to that in WT mice. The number of doublecortin (DCX) and neuronal nuclei (NeuN)-positive cells in HDG was decreased after photothrombotic ischemia in TLR2 KO mice compared to that in WT mice. The survival rate of cells in HDG was decreased in TLR2 KO mice compared to that in WT mice. In contrast, the number of cleaved-caspase 3 (apoptotic marker) and the number of GFAP (glia marker)/BrdU double-positive cells in TLR2 KO mice were higher than that in WT mice. These results suggest that TLR2 can promote adult neurogenesis from neural stem cell of hippocampal dentate gyrus through increasing proliferation, differentiation, and survival from neural stem cells after ischemic injury of the brain.

Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice

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Sun Wei-Zen

2008-09-01

Full Text Available Abstract Background Mice lacking the preproenkephalin (ppENK gene are hyperalgesic and show more anxiety and aggression than wild-type (WT mice. The marked behavioral changes in ppENK knock-out (KO mice appeared to occur in supraspinal response to painful stimuli. However the functional role of enkephalins in the supraspinal nociceptive processing and their underlying mechanism is not clear. The aim of present study was to compare supraspinal nociceptive and morphine antinociceptive responses between WT and ppENK KO mice. Results The genotypes of bred KO mice were confirmed by PCR. Met-enkephalin immunoreactive neurons were labeled in the caudate-putamen, intermediated part of lateral septum, lateral globus pallidus, intermediated part of lateral septum, hypothalamus, and amygdala of WT mice. Met-enkephalin immunoreactive neurons were not found in the same brain areas in KO mice. Tail withdrawal and von Frey test results did not differ between WT and KO mice. KO mice had shorter latency to start paw licking than WT mice in the hot plate test. The maximal percent effect of morphine treatments (5 mg/kg and 10 mg/kg, i.p. differed between WT and KO mice in hot plate test. The current source density (CSD profiles evoked by peripheral noxious stimuli in the primary somatosenstory cortex (S1 and anterior cingulate cortex (ACC were similar in WT and KO mice. After morphine injection, the amplitude of the laser-evoked sink currents was decreased in S1 while the amplitude of electrical-evoked sink currents was increased in the ACC. These differential morphine effects in S1 and ACC were enhanced in KO mice. Facilitation of synaptic currents in the ACC is mediated by GABA inhibitory interneurons in the local circuitry. Percent increases in opioid receptor binding in S1 and ACC were 5.1% and 5.8%, respectively. Conclusion The present results indicate that the endogenous enkephalin system is not involved in acute nociceptive transmission in the spinal cord

Contribution of PPARγ in modulation of acrolein-induced inflammatory signaling in gp91phox knock-out mice.

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Yousefipour, Zivar; Chug, Neha; Marek, Katarzyna; Nesbary, Alicia; Mathew, Joseph; Ranganna, Kasturi; Newaz, Mohammad A

2017-08-01

Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91 phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass) -1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass) -1 ·day -1 , orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.

Involvement of interleukin-1 in lead nitrate-induced hypercholesterolemia in mice.

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Kojima, Misaki; Ashino, Takashi; Yoshida, Takemi; Iwakura, Yoichiro; Degawa, Masakuni

2012-01-01

Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cholesterol 7α-hydroxylase (Cyp7a1) are rate-limiting enzymes for cholesterol biosynthesis and catabolism, respectively. Involvement of inflammatory cytokines, particularly interleukin-1 (IL-1), in alterations of HMGR and Cyp7a1 gene expression during development of lead nitrate (LN)-induced hypercholesterolemia was examined in IL-1α/β-knockout (IL-1-KO) and wild-type (WT) mice. Lead nitrate treatment of WT mice led to not only a marked downregulation of the Cyp7a1 gene at 6-12 h, but also a significant upregulation of the HMGR gene at 12 h. However, such changes were not observed at significant levels in IL-1-KO mice, although a slight, transient downregulation of the Cyp7a1 gene and a minimal upregulation of the HMGR gene occurred at 6 h and 24 h, respectively. Consequently, LN treatment led to development of hypercholesterolemia at 24 h in WT mice, but not in IL-1-KO mice. Furthermore, in WT mice, significant LN-mediated increases were observed at 3-6 h in hepatic IL-1 levels, which can modulate gene expression of Cyp7a1 and HMGR. These findings indicate that, in mice, LN-mediated increases in hepatic IL-1 levels contribute, at least in part, to altered expressions of Cyp7a1 and HMGR genes, and eventually to hypercholesterolemia development.

Relationship between Sustained Reductions in Plasma Lipid and Lipoprotein Concentrations with Apheresis and Plasma Levels and mRNA Expression of PTX3 and Plasma Levels of hsCRP in Patients with HyperLp(a)lipoproteinemia

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Stefanutti, Claudia; Mazza, Fabio; Steiner, Michael; Watts, Gerald F.; De Nève, Joel; Pasqualetti, Daniela; Paal, Juergen

2016-01-01

The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI) (LA) on plasma levels of pentraxin 3 (PTX3), an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(a)lipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged 55 ± 9.3 years (mean ± SD), were enrolled in the study. PTX3 soluble protein levels in plasma were unchanged by 2 sessions of LA; however, a downregulation of mRNA expression for PTX3 was observed, starting with the first session of LA (p < 0.001). The observed reduction was progressively increased in the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session. A statistically significantly greater treatment-effect correlation was observed in patients undergoing weekly treatments, compared with those undergoing treatment every 15 days. A progressive reduction in plasma levels of C-reactive protein was also seen from the first session of LA, with a statistically significant linear correlation for treatment-effect in the change in plasma levels of this established inflammatory marker (R 2 = 0.99; p < 0.001). Our findings suggest that LA has anti-inflammatory and endothelium protective effects beyond its well-established efficacy in lowering apoB100-containing lipoproteins. PMID:26903710

Desacyl Ghrelin Decreases Anxiety-like Behavior in Male Mice.

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Mahbod, Parinaz; Smith, Eric P; Fitzgerald, Maureen E; Morano, Rachel L; Packard, Benjamin A; Ghosal, Sriparna; Scheimann, Jessie R; Perez-Tilve, Diego; Herman, James P; Tong, Jenny

2018-01-01

Ghrelin is a 28-amino acid polypeptide that regulates feeding, glucose metabolism, and emotionality (stress, anxiety, and depression). Plasma ghrelin circulates as desacyl ghrelin (DAG) or, in an acylated form, acyl ghrelin (AG), through the actions of ghrelin O-acyltransferase (GOAT), exhibiting low or high affinity, respectively, for the growth hormone secretagogue receptor (GHSR) 1a. We investigated the role of endogenous AG, DAG, and GHSR1a signaling on anxiety and stress responses using ghrelin knockout (Ghr KO), GOAT KO, and Ghsr stop-floxed (Ghsr null) mice. Behavioral and hormonal responses were tested in the elevated plus maze and light/dark (LD) box. Mice lacking both AG and DAG (Ghr KO) increased anxiety-like behaviors across tests, whereas anxiety reactions were attenuated in DAG-treated Ghr KO mice and in mice lacking AG (GOAT KO). Notably, loss of GHSR1a (Ghsr null) did not affect anxiety-like behavior in any test. Administration of AG and DAG to Ghr KO mice with lifelong ghrelin deficiency reduced anxiety-like behavior and decreased phospho-extracellular signal-regulated kinase phosphorylation in the Edinger-Westphal nucleus in wild-type mice, a site normally expressing GHSR1a and involved in stress- and anxiety-related behavior. Collectively, our data demonstrate distinct roles for endogenous AG and DAG in regulation of anxiety responses and suggest that the behavioral impact of ghrelin may be context dependent. Copyright © 2018 Endocrine Society.

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

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Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A

2013-02-15

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

Role of Keap1-Nrf2 signaling in depression and dietary intake of glucoraphanin confers stress resilience in mice

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Yao, Wei; Zhang, Ji-chun; Ishima, Tamaki; Dong, Chao; Yang, Chun; Ren, Qian; Ma, Min; Han, Mei; Wu, Jin; Suganuma, Hiroyuki; Ushida, Yusuke; Yamamoto, Masayuki; Hashimoto, Kenji

2016-01-01

The transcription factor Keap1-Nrf2 system plays a key role in inflammation which is involved in depression. We found lower expression of Keap1 and Nrf2 proteins in the prefrontal cortex (PFC), CA3 and dentate gyrus (DG) of hippocampus in mice with depression-like phenotype compared to control mice. Serum levels of pro-inflammatory cytokines in Nrf2 knock-out (KO) mice were higher than those of wild-type mice, suggestive of enhanced inflammation in KO mice. Decreased brain-derived neurotrophi...

A conditioned aversion study of sucrose and SC45647 taste in TRPM5 knockout mice.

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Eddy, Meghan C; Eschle, Benjamin K; Peterson, Darlene; Lauras, Nathan; Margolskee, Robert F; Delay, Eugene R

2012-06-01

Previously, published studies have reported mixed results regarding the role of the TRPM5 cation channel in signaling sweet taste by taste sensory cells. Some studies have reported a complete loss of sweet taste preference in TRPM5 knockout (KO) mice, whereas others have reported only a partial loss of sweet taste preference. This study reports the results of conditioned aversion studies designed to motivate wild-type (WT) and KO mice to respond to sweet substances. In conditioned taste aversion experiments, WT mice showed nearly complete LiCl-induced response suppression to sucrose and SC45647. In contrast, TRPM5 KO mice showed a much smaller conditioned aversion to either sweet substance, suggesting a compromised, but not absent, ability to detect sweet taste. A subsequent conditioned flavor aversion experiment was conducted to determine if TRPM5 KO mice were impaired in their ability to learn a conditioned aversion. In this experiment, KO and WT mice were conditioned to a mixture of SC45647 and amyl acetate (an odor cue). Although WT mice avoided both components of the stimulus mixture, they avoided SC45647 more than the odor cue. The KO mice also avoided both stimuli, but they avoided the odor component more than SC45647, suggesting that while the KO mice are capable of learning an aversion, to them the odor cue was more salient than the taste cue. Collectively, these findings suggest the TRPM5 KO mice have some residual ability to detect SC45647 and sucrose, and, like bitter, there may be a TRPM5-independent transduction pathway for detecting these substances.

Prophylactic administration of an extract from Plantaginis Semen and its major component aucubin inhibits mechanical allodynia caused by paclitaxel in mice

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Tsugunobu Andoh

2016-07-01

Full Text Available The chemotherapeutic agent paclitaxel (PTX causes peripheral neuropathy as a major dose-limiting side effect, and this peripheral neuropathy is difficult to control. Our previous report showed that prophylactic repetitive administration of goshajinkigan (牛車腎氣丸 niú chē shèn qì wán, but not hachimijiogan (八味地黃丸 bā wèi dì huáng wán, which lacks two of the constituents of goshajinkigan, inhibited PTX-induced mechanical allodynia in mice. Thus, the herbal medicines Plantaginis Semen (車前子 chē qián zǐ or Achyranthis Radix (牛膝 niú xī may contribute to the inhibitory action of goshajinkigan on the exacerbation of PTX-induced mechanical allodynia [Andoh et al, J. Tradit. Complement. Med. 2014; 4: 293–297]. Therefore, in this study, we examined whether an extract of Plantaginis Semen (EPS or Achyranthis Radix (EAR would relieve PTX-induced mechanical allodynia in mice. A single intraperitoneal injection of PTX caused mechanical allodynia, which peaked on day 14 after injection. Repetitive oral administration of EPS, but not EAR, starting from the day after PTX injection significantly inhibited the exacerbation of PTX-induced mechanical allodynia. Repetitive intraperitoneal injection of aucubin, one of the main components of EPS, starting from the day after PTX injection also significantly reduced PTX-induced mechanical allodynia. However, repetitive intraperitoneal injection of geniposide acid (a precursor of aucubin or catalpol (a metabolite of aucubin did not prevent the exacerbation of mechanical allodynia. These results suggest that prophylactic administration of EPS is effective for preventing the exacerbation of PTX-induced allodynia. Aucubin may contribute to the inhibitory action of EPS on the exacerbation of PTX-induced allodynia.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

International Nuclear Information System (INIS)

Matsumura, Kaori; Taketomi, Takaharu; Yoshizaki, Keigo; Arai, Shinsaku; Sanui, Terukazu; Yoshiga, Daigo; Yoshimura, Akihiko; Nakamura, Seiji

2011-01-01

Research highlights: → Sprouty2-deficient mice exhibit cleft palate as a result of failure of palatal shelf elevation. → We examined palate cell proliferation in Sprouty2-deficient mice. → Palate mesenchymal cell proliferation was increased in Sprouty2 KO mice. → Sprouty2 plays roles in murine palatogenesis by regulating cell proliferation. -- Abstract: Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

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Matsumura, Kaori [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Taketomi, Takaharu, E-mail: taketomi@dent.kyushu-u.ac.jp [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshizaki, Keigo [Section of Orthodontics, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Arai, Shinsaku [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Sanui, Terukazu [Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshiga, Daigo [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshimura, Akihiko [Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Japan Science and Technology Agency (JST), CREST, Chiyoda-ku, Tokyo 102-0075 (Japan); Nakamura, Seiji [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2011-01-28

Research highlights: {yields} Sprouty2-deficient mice exhibit cleft palate as a result of failure of palatal shelf elevation. {yields} We examined palate cell proliferation in Sprouty2-deficient mice. {yields} Palate mesenchymal cell proliferation was increased in Sprouty2 KO mice. {yields} Sprouty2 plays roles in murine palatogenesis by regulating cell proliferation. -- Abstract: Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

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Nahampun, Hartinio N; López-Arredondo, Damar; Xu, Xing; Herrera-Estrella, Luis; Wang, Kan

2016-05-01

Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation

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Reno, Candace M.; Puente, Erwin C.; Sheng, Zhenyu; Daphna-Iken, Dorit; Bree, Adam J.; Routh, Vanessa H.; Kahn, Barbara B.

2017-01-01

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose. PMID:27797912

Western Diet-Induced Dysbiosis in Farnesoid X Receptor Knockout Mice Causes Persistent Hepatic Inflammation after Antibiotic Treatment.

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Jena, Prasant K; Sheng, Lili; Liu, Hui-Xin; Kalanetra, Karen M; Mirsoian, Annie; Murphy, William J; French, Samuel W; Krishnan, Viswanathan V; Mills, David A; Wan, Yu-Jui Yvonne

2017-08-01

Patients who have liver cirrhosis and liver cancer also have reduced farnesoid X receptor (FXR). The current study analyzes the effect of diet through microbiota that affect hepatic inflammation in FXR knockout (KO) mice. Wild-type and FXR KO mice were on a control (CD) or Western diet (WD) for 10 months. In addition, both CD- and WD-fed FXR KO male mice, which had hepatic lymphocyte and neutrophil infiltration, were treated by vancomycin, polymyxin B, and Abx (ampicillin, neomycin, metronidazole, and vancomycin). Mice were subjected to morphological analysis as well as gut microbiota and bile acid profiling. Male WD-fed FXR KO mice had the most severe steatohepatitis. FXR KO also had reduced Firmicutes and increased Proteobacteria, which could be reversed by Abx. In addition, Abx eliminated hepatic neutrophils and lymphocytes in CD-fed, but not WD-fed, FXR KO mice. Proteobacteria and Bacteroidetes persisted in WD-fed FXR KO mice even after Abx treatment. Only polymyxin B could reduce hepatic lymphocytes in WD-fed FXR KO mice. The reduced hepatic inflammation by antibiotics was accompanied by decreased free and conjugated secondary bile acids as well as changes in gut microbiota. Our data revealed that Lactococcus, Lactobacillus, and Coprococcus protect the liver from inflammation. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Large-conductance Ca2+-activated K+ channel β1-subunit knockout mice are not hypertensive

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Garver, Hannah; Galligan, James J.; Fink, Gregory D.

2011-01-01

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca2+ channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension. PMID:21131476

Differential Impact of miR-21 on Pain and Associated Affective and Cognitive Behavior after Spared Nerve Injury in B7-H1 ko Mouse

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Franziska Karl

2017-07-01

Full Text Available MicroRNAs (miRNAs are increasingly recognized as regulators of immune and neuronal gene expression and are potential master switches in neuropathic pain pathophysiology. miR-21 is a promising candidate that may link the immune and the pain system. To investigate the pathophysiological role of miR-21 in neuropathic pain, we assessed mice deficient of B7 homolog 1 (B7-H1, a major inhibitor of inflammatory responses. In previous studies, an upregulation of miR-21 had been shown in mouse lymphocytes. Young (8 weeks, middle-aged (6 months, and old (12 months B7-H1 ko mice and wildtype littermates (WT received a spared nerve injury (SNI. We assessed thermal withdrawal latencies and mechanical withdrawal thresholds. Further, we performed tests for anxiety-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, and dorsal root ganglia (DRG at distinct time points after SNI. We found mechanical hyposensitivity with increasing age of naïve B7-H1 ko mice. Young and middle-aged B7-H1 ko mice were more sensitive to mechanical stimuli compared to WT mice (young: p < 0.01, middle-aged: p < 0.05. Both genotypes developed mechanical and heat hypersensitivity (p < 0.05 after SNI, without intergroup differences. No relevant differences were found after SNI in three tests for anxiety like behavior in B7-H1 ko and WT mice. Also, SNI had no effect on cognition. B7-H1 ko and WT mice showed a higher miR-21 expression (p < 0.05 and invasion of macrophages and T cells in the injured nerve 7 days after SNI without intergroup differences. Our study reveals that increased miR-21 expression in peripheral nerves after SNI is associated with reduced mechanical and heat withdrawal thresholds. These results point to a role of miR-21 in the pathophysiology of neuropathic pain, while affective behavior and cognition seem to be spared. Contrary to expectations, B7-H1 ko mice did not show higher miR-21

Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

Institute of Scientific and Technical Information of China (English)

Shengqiang Chen; Xuegang Luo; Quan Yang; Weiwen Sun; Kaiyi Cao; Xi Chen; Yueling Huang; Lijun Dai; Yonghong Yi

2011-01-01

In the present study, Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome. The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts, indicating a learning and memory disorder. After treatment with 30, 60, 90, 120, or 200 mg/kg lithium chloride, the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated, in particular, the 200 mg/kg lithium chloride treatment had the most significant effect. Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta, an inactive form of glycogen synthase kinase 3 beta, in the cerebral cortex and hippocampus of the Fmr1 KO mice. These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice, possibly by inhibiting glycogen synthase kinase 3 beta activity.

No cytotoxic effects from application of pentoxifylline to spermatozoa on subsequent pre-implantation embryo development in mice

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Mohammad Ali Khalili

2017-06-01

Full Text Available The aim was to assess the effect of spermatozoa exposed to PTX on the rates of fertilization and embryo development and apoptotic cells within blastocysts in an animal model. Mice Oocytes were inseminated with spermatozoa exposed to 3.6 mmol PTX for 30 min, or with neat spermatozoa. Then fertilization and embryo development rate, blastocyst formation and quality, as well as total cell number of blastocyst, and DNA fragmentation index (DFI in blastocysts were surveyed in both groups. Fertilization and embryo development rate were similar between the groups. The rates of blastocyst formation did not differ significantly between control and PTX groups (52.4% vs. 51.8%. The average of total cell count in blastocysts and DFI in control and PTX groups were also insignificant (31.08 ± 1.5 vs. 34.14 ± 1.5 and 9.76 ± 5.0 vs. 11.77 ± 5.4. Application of PTX for enhancing sperm motility does not cause a cytotoxic effect on subsequent embryo development and embryo genome integrity.

Effect of adiponectin deficiency on intestinal damage and hematopoietic responses of mice exposed to gamma radiation

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Ponemone, Venkatesh; Fayad, Raja; Gove, Melissa E.; Pini, Maria [Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL 60612 (United States); Fantuzzi, Giamila, E-mail: giamila@uic.edu [Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL 60612 (United States)

2010-08-07

Adiponectin (APN) is an adipose tissue-derived cytokine that regulates insulin sensitivity and inflammation. It is also involved in modulation of cell proliferation by binding to various growth factors. Based on its known effects in modulating cell proliferation and oxidative stress, APN may potentially be involved in regulating tissue damage and repair following irradiation. Adiponectin KO mice and their WT littermates were exposed to a single whole-body dose of 3 or 6 Gy gamma radiation. Radiation-induced alterations were studied in jejunum, blood, bone marrow and thymus at days 1 and 5 post-irradiation and compared with sham-irradiated groups. In WT mice, irradiation did not significantly alter serum APN levels while inducing a significant decrease in serum leptin. Irradiation caused a significant reduction in thymocyte cellularity, with concomitant decrease in CD4{sup +}, CD8{sup +} and CD4{sup +}CD8{sup +} T cell populations, with no significant differences between WT and APN KO mice. Irradiation resulted in a significantly higher increase in the frequency of micronucleated reticulocytes in the blood of APN KO compared with WT mice, whereas frequency of micronucleated normochromatic erythrocytes in the bone marrow at day 5 was significantly higher in WT compared with APN KO mice. Finally, irradiation induced similar alterations in villus height and crypt cell proliferation in the jejunum of WT and APN KO mice. Jejunum explants from sham-irradiated APN KO mice produced higher levels of IL-6 compared with tissue from WT animals, but the difference was no longer apparent following irradiation. Our data indicate that APN deficiency does not play a significant role in modulating radiation-induced gastrointestinal injury in mice, while it may participate in regulation of damage to the hematopoietic system.

Effect of adiponectin deficiency on intestinal damage and hematopoietic responses of mice exposed to gamma radiation

International Nuclear Information System (INIS)

Ponemone, Venkatesh; Fayad, Raja; Gove, Melissa E.; Pini, Maria; Fantuzzi, Giamila

2010-01-01

Adiponectin (APN) is an adipose tissue-derived cytokine that regulates insulin sensitivity and inflammation. It is also involved in modulation of cell proliferation by binding to various growth factors. Based on its known effects in modulating cell proliferation and oxidative stress, APN may potentially be involved in regulating tissue damage and repair following irradiation. Adiponectin KO mice and their WT littermates were exposed to a single whole-body dose of 3 or 6 Gy gamma radiation. Radiation-induced alterations were studied in jejunum, blood, bone marrow and thymus at days 1 and 5 post-irradiation and compared with sham-irradiated groups. In WT mice, irradiation did not significantly alter serum APN levels while inducing a significant decrease in serum leptin. Irradiation caused a significant reduction in thymocyte cellularity, with concomitant decrease in CD4 + , CD8 + and CD4 + CD8 + T cell populations, with no significant differences between WT and APN KO mice. Irradiation resulted in a significantly higher increase in the frequency of micronucleated reticulocytes in the blood of APN KO compared with WT mice, whereas frequency of micronucleated normochromatic erythrocytes in the bone marrow at day 5 was significantly higher in WT compared with APN KO mice. Finally, irradiation induced similar alterations in villus height and crypt cell proliferation in the jejunum of WT and APN KO mice. Jejunum explants from sham-irradiated APN KO mice produced higher levels of IL-6 compared with tissue from WT animals, but the difference was no longer apparent following irradiation. Our data indicate that APN deficiency does not play a significant role in modulating radiation-induced gastrointestinal injury in mice, while it may participate in regulation of damage to the hematopoietic system.

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice.

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Keith Dadson

Full Text Available Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM remodeling in response to pressure overload (PO. Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO and wild-type (WT mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson's trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects.

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

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Xing, Lianping; Chen, Di; Boyce, Brendan F.

2013-01-01

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth...

Trpc2-deficient lactating mice exhibit altered brain and behavioral responses to bedding stimuli.

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Hasen, Nina S; Gammie, Stephen C

2011-03-01

The trpc2 gene encodes an ion channel involved in pheromonal detection and is found in the vomeronasal organ. In tprc2(-/-) knockout (KO) mice, maternal aggression (offspring protection) is impaired and brain Fos expression in females in response to a male are reduced. Here we examine in lactating wild-type (WT) and KO mice behavioral and brain responses to different olfactory/pheromonal cues. Consistent with previous studies, KO dams exhibited decreased maternal aggression and nest building, but we also identified deficits in nighttime nursing and increases in pup weight. When exposed to the bedding tests, WT dams typically ignored clean bedding, but buried male-soiled bedding from unfamiliar males. In contrast, KO dams buried both clean and soiled bedding. Differences in brain Fos expression were found between WT and KO mice in response to either no bedding, clean bedding, or soiled bedding. In the accessory olfactory bulb, a site of pheromonal signal processing, KO mice showed suppressed Fos activation in the anterior mitral layer relative to WT mice in response to clean and soiled bedding. However, in the medial and basolateral amygdala, KO mice showed a robust Fos response to bedding, suggesting that regions of the amygdala canonically associated with pheromonal sensing can be active in the brains of KO mice, despite compromised signaling from the vomeronasal organ. Together, these results provide further insights into the complex ways by which pheromonal signaling regulates the brain and behavior of the maternal female. Copyright © 2010 Elsevier B.V. All rights reserved.

Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

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Satoko eHattori

2012-10-01

Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

Dopamine D5 receptor modulates male and female sexual behavior in mice.

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Kudwa, A E; Dominguez-Salazar, E; Cabrera, D M; Sibley, D R; Rissman, E F

2005-07-01

Dopamine exerts its actions through at least five receptor (DAR) isoforms. In female rats, D5 DAR may be involved in expression of sexual behavior. We used a D5 knockout (D5KO) mouse to assess the role of D5 DAR in mouse sexual behavior. Both sexes of D5KO mice are fertile and exhibit only minor disruptions in exploratory locomotion, startle, and prepulse inhibition responses. This study was conducted to characterize the sexual behavior of male and female D5KO mice relative to their WT littermates. Female WT and D5KO littermates were ovariectomized and given a series of sexual behavior tests after treatment with estradiol benzoate (EB) and progesterone (P). Once sexual performance was optimal the dopamine agonist, apomorphine (APO), was substituted for P. Male mice were observed in pair- and trio- sexual behavior tests. To assess whether the D5 DAR is involved in rewarding aspects of sexual behavior, WT and D5KO male mice were tested for conditioned place preference. Both WT and D5KO females can display receptivity after treatment with EB and P, but APO was only able to facilitate receptivity in EB-primed WT, not in D5KO, mice. Male D5KO mice display normal masculine sexual behavior in mating tests. In conditioned preference tests, WT males formed a conditioned preference for context associated with either intromissions alone or ejaculation as the unconditioned stimulus. In contrast, D5KO males only showed a place preference when ejaculation was paired with the context. In females, the D5 DAR is essential for the actions of dopamine on receptivity. In males, D5 DAR influences rewarding aspects of intromissions. Taken together, the work suggests that the D5 receptor mediates dopamine's action on sexual behavior in both sexes, perhaps via a reward pathway.

Kynurenine 3-monooxygenase is implicated in antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions.

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Tashiro, Tomoyuki; Murakami, Yuki; Mouri, Akihiro; Imamura, Yukio; Nabeshima, Toshitaka; Yamamoto, Yasuko; Saito, Kuniaki

2017-01-15

l-Tryptophan (TRP) is metabolized via serotonin and kynurenine pathways (KP). Several studies have demonstrated that abnormality of both pathways is involved in the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the KP, has been suggested to play major roles in physiological and pathological events mediated by bioactive kynurenine metabolites. In this study, we investigated the role of KMO in the emotional and cognitive functions by using KMO knockout (KO) mice. We measured contents of TRP and monoamines and their metabolites in the serum and hippocampus of KMO KO mice. Further, we investigated whether antidepressants improved the depressive-like behaviors in KMO KO mice. KMO KO mice showed depressive-like behaviors such as decreased sucrose preference and increased immobility in the forced swimming test and high anxiety by decreased time spent in the center area of open field. But, there was no difference in spontaneous alternation in Y-maze test, counts of rearing or locomotor activity. Higher contents of TRP metabolites such as kynurenine (KYN), kynurenic acid (KA), anthranilic acid (AA), and 3-hydroxykynurenine (3-HK) in the serum and hippocampus and decreased serotonin turnover and higher content of normetanephrine (NM) in the hippocampus were observed in the KMO KO mice. Although both antidepressant attenuated increase of immobility, sertraline but not imipramine improved decrease of sucrose preference in the KMO KO mice. These findings suggested that KMO KO mice show antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions via abnormality of kynurenine metabolism with good validities as MDD model. Copyright © 2016 Elsevier B.V. All rights reserved.

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

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Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

2014-06-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

Hyperactivity of Hypothalamic-Pituitary-Adrenal Axis Due to Dysfunction of the Hypothalamic Glucocorticoid Receptor in Sigma-1 Receptor Knockout Mice

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Tingting Di

2017-09-01

Full Text Available Sigma-1 receptor knockout (σ1R-KO mice exhibit a depressive-like phenotype. Because σ1R is highly expressed in the neuronal cells of hypothalamic paraventricular nuclei (PVN, this study investigated the influence of σ1R deficiency on the regulation of the hypothalamic-pituitary-adrenocortical (HPA axis. Here, we show that the levels of basal serum corticosterone (CORT, adrenocorticotropic hormone (ACTH and corticotrophin releasing factor (CRF as well as the level of CRF mRNA in PVN did not significantly differ between adult male σ1R-KO mice and wild-type (WT mice. Acute mild restraint stress (AMRS induced a higher and more sustainable increase in activity of HPA axis and CRF expression in σ1R-KO mice. Percentage of dexamethasone (Dex-induced reduction in level of CORT was markedly attenuated in σ1R−/− mice. The levels of glucocorticoid receptor (GR and protein kinase C (PKC phosphorylation were reduced in the PVN of σ1R-KO mice and σ1R antagonist NE100-treated WT mice. The exposure to AMRS in σ1R-KO mice induced a stronger phosphorylation of cAMP-response element binding protein (CREB in PVN than that in WT mice. Intracerebroventricular (i.c.v. injection of PKC activator PMA for 3 days in σ1R-KO mice not only recovered the GR phosphorylation and the percentage of Dex-reduced CORT but also corrected the AMRS-induced hyperactivity of HPA axis and enhancement of CRF mRNA and CREB phosphorylation. Furthermore, the injection (i.c.v. of PMA in σ1R-KO mice corrected the prolongation of immobility time in forced swim test (FST and tail suspension test (TST. These results indicate that σ1R deficiency causes down-regulation of GR by reducing PKC phosphorylation, which attenuates GR-mediated feedback inhibition of HPA axis and facilitates the stress response of HPA axis leading to the production of depressive-like behaviors.

Loss of PDZK1 causes coronary artery occlusion and myocardial infarction in Paigen diet-fed apolipoprotein E deficient mice.

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Ayce Yesilaltay

2009-12-01

Full Text Available PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI, and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO mice are characterized by a marked reduction of SR-BI protein expression ( approximately 95% in the liver (lesser or no reduction in other organs with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol ('Western' diet-fed murine apolipoprotein E (apoE KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI.Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic 'Paigen' diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted, were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle.These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.

The metabotropic glutamate 5 receptor modulates extinction and reinstatement of methamphetamine-seeking in mice.

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Rose Chesworth

Full Text Available Methamphetamine (METH is a highly addictive psychostimulant with no therapeutics registered to assist addicts in discontinuing use. Glutamatergic dysfunction has been implicated in the development and maintenance of addiction. We sought to assess the involvement of the metabotropic glutamate 5 receptor (mGlu5 in behaviours relevant to METH addiction because this receptor has been implicated in the actions of other drugs of abuse, including alcohol, cocaine and opiates. mGlu5 knockout (KO mice were tested in intravenous self-administration, conditioned place preference and locomotor sensitization. Self-administration of sucrose was used to assess the response of KO mice to a natural reward. Acquisition and maintenance of self-administration, as well as the motivation to self-administer METH was intact in mGlu5 KO mice. Importantly, mGlu5 KO mice required more extinction sessions to extinguish the operant response for METH, and exhibited an enhanced propensity to reinstate operant responding following exposure to drug-associated cues. This phenotype was not present when KO mice were tested in an equivalent paradigm assessing operant responding for sucrose. Development of conditioned place preference and locomotor sensitization were intact in KO mice; however, conditioned hyperactivity to the context previously paired with drug was elevated in KO mice. These data demonstrate a role for mGlu5 in the extinction and reinstatement of METH-seeking, and suggests a role for mGlu5 in regulating contextual salience.

Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway.

Science.gov (United States)

Chen, Dongshao; Lin, Xiaoting; Zhang, Cheng; Liu, Zhentao; Chen, Zuhua; Li, Zhongwu; Wang, Jingyuan; Li, Beifang; Hu, Yanting; Dong, Bin; Shen, Lin; Ji, Jiafu; Gao, Jing; Zhang, Xiaotian

2018-01-26

Paclitaxel (PTX) is widely used in the front-line chemotherapy for gastric cancer (GC), but resistance limits its use. Due to the lack of proper models, mechanisms underlying PTX resistance in GC were not well studied. Using established PTX-resistant GC cell sublines HGC-27R, we for the first time integrated biological traits and molecular mechanisms of PTX resistance in GC. Data revealed that PTX-resistant GC cells were characterized by microtubular disorders, an EMT phenotype, reduced responses to antimitotic drugs, and resistance to apoptosis (marked by upregulated β-tubulin III, vimentin, attenuated changes in G 2 /M molecules or pro-apoptotic factors in response to antimitotic drugs or apoptotic inducers, respectively). Activation of the phosphoinositide 3-kinase, the serine/threonine kinase Akt and mammalian target of rapamycin (PI3K/Akt/mTOR) and mitogen-activated protein kinase (MAPK) pathways were also observed, which might be the reason for above phenotypic alternations. In vitro data suggested that targeting these pathways were sufficient to elicit antitumor responses in PTX-resistant GC, in which the dual PI3K/mTOR inhibitor BEZ235 displayed higher therapeutic efficiency than the mTOR inhibitor everolimus or the MEK inhibitor AZD6244. Antitumor effects of BEZ235 were also confirmed in mice bearing HGC-27R tumors. Thus, these data suggest that PI3K/Akt/mTOR and MAPK pathway inhibition, especially PI3K/mTOR dual blockade, might be a promising therapeutic strategy against PTX-resistant GC.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation.

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Reno, Candace M; Puente, Erwin C; Sheng, Zhenyu; Daphna-Iken, Dorit; Bree, Adam J; Routh, Vanessa H; Kahn, Barbara B; Fisher, Simon J

2017-03-01

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose. © 2017 by the American Diabetes Association.

Ethanol self-administration in serotonin transporter knockout mice: unconstrained demand and elasticity.

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Lamb, R J; Daws, L C

2013-10-01

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self-administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self-administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self-administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration

DEFF Research Database (Denmark)

Nunes, Ana Filipa; Montero, Maria; Franquinho, Filipa

2009-01-01

Transthyretin (TTR) has been regarded as a neuroprotective protein given that TTR knockout (KO) mice display increased susceptibility for amyloid beta deposition and memory deficits during aging. In parallel, TTR KO mice have increased levels of neuropeptide Y (NPY), which promotes neuroprotectio...

Germinated Brown Rice Attenuates Atherosclerosis and Vascular Inflammation in Low-Density Lipoprotein Receptor-Knockout Mice.

Science.gov (United States)

Zhao, Ruozhi; Ghazzawi, Nora; Wu, Jiansu; Le, Khuong; Li, Chunyang; Moghadasian, Mohammed H; Siow, Yaw L; Apea-Bah, Franklin B; Beta, Trust; Yin, Zhengfeng; Shen, Garry X

2018-05-02

The present study investigates the impact of germinated brown rice (GBR) on atherosclerosis and the underlying mechanism in low-density lipoprotein receptor-knockout (LDLr-KO) mice. The intensity of atherosclerosis in aortas of LDLr-KO mice receiving diet supplemented with 60% GBR (weight/weight) was significantly less than that in mice fed with 60% white rice (WR) or control diet ( p mice fed with WR diet was significantly more than that from mice receiving the control diet ( p mice in comparison to the WR diet ( p mice compared to WR. The anti-atherosclerotic effect of GBR in LDLr-KO mice at least in part results from its anti-inflammatory activity.

Interaction of organic cation transporter 3 (SLC22A3) and amphetamine.

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Zhu, Hao-Jie; Appel, David I; Gründemann, Dirk; Markowitz, John S

2010-07-01

The organic cation transporter (OCT) 3 is widely expressed in various organs in humans, and involved in the disposition of many exogenous and endogenous compounds. Several lines of evidence have suggested that OCT3 expressed in the brain plays an important role in the regulation of neurotransmission. Relative to wild-type (WT) animals, Oct3 knockout (KO) mice have displayed altered behavioral and neurochemical responses to psychostimulants such as amphetamine (AMPH) and methamphetamine. In the present study, both in vitro and in vivo approaches were utilized to explore potential mechanisms underlying the disparate neuropharmacological effects observed following AMPH exposure in Oct3 KO mice. In vitro uptake studies conducted in OCT3 transfected cells indicated that dextroamphetamine (d-AMPH) is not a substrate of OCT3. However, OCT3 was determined to be a high-capacity and low-affinity transporter for the neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5-HT). Inhibition studies demonstrated that d-AMPH exerts relatively weak inhibitory effects on the OCT3-mediated uptake of DA, NE, 5-HT, and the model OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide. The IC(50) values were determined to be 41.5 +/- 7.5 and 24.1 +/- 7.0 microM for inhibiting DA and 5-HT uptake, respectively, while 50% inhibition of NE and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide uptake was not achieved by even the highest concentration of d-AMPH applied (100 microM). Furthermore, the disposition of d-AMPH in various tissues including the brain, liver, heart, kidney, muscle, intestine, spleen, testis, uterus, and plasma were determined in both male and female Oct3 KO and WT mice. No significant difference was observed between either genotypes or sex in all tested organs and tissues. Our findings suggest that OCT3 is not a prominent factor influencing the disposition of d-AMPH. Additionally, based upon the inhibitory potency observed in vitro, d

Male mice with deleted Wolframin (Wfs1 gene have reduced fertility

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Aunapuu Marina

2009-08-01

Full Text Available Abstract Background Wolfram Syndrome (WS is an autosomal recessive disorder characterised by non-autoimmune diabetes mellitus, optic atrophy, cranial diabetes insipidus and sensorineural deafness. Some reports have described hypogonadism in male WS patients. The aim of our study was to find out whether Wfs1 deficient (Wfs1KO male mice have reduced fertility and, if so, to examine possible causes. Methods Wfs1KO mice were generated by homologous recombination. Both Wfs1KO and wild type (wt male mice were mated with wt female mice. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analysed. Serum testosterone and FSH concentrations were also measured. Results The pregnancy rate in wt females mated with Wfs1KO males was significantly lower than in the control group (15% vs. 32%; p Conclusion The impaired fertility of Wfs1KO male mice is most likely due to changes in sperm morphology and reduced number of spermatogenic cells. The exact mechanism through which the Wfs1 gene influences sperm morphology needs to be clarified in further studies.

A distinctive patchy osteomalacia characterises Phospho1-deficient mice.

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Boyde, Alan; Staines, Katherine A; Javaheri, Behzad; Millan, Jose Luis; Pitsillides, Andrew A; Farquharson, Colin

2017-08-01

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase

Sex differences in insulin resistance in GABAB1 knockout mice.

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Bonaventura, M M; Rodriguez, D; Ferreira, M L; Crivello, M; Repetto, E M; Bettler, B; Libertun, C; Lux-Lantos, V A

2013-02-27

We have previously demonstrated that the absence of functional GABA B receptors (GABABRs) disturbs glucose homeostasis in GABAB1KO mice. The aim of this work was to extend our studies of these alterations in GABAB1KO mice and investigate the sexual differences therein. Male and female, GABAB1KO and WT mice were used. Glucose and insulin tolerance tests (GTT and ITT), and insulin and glucagon secretion tests (IST and GST) were performed. Blood glucose, serum insulin and hyperglycemic hormones were determined, and HOMA-IR calculated. Skeletal muscle insulin receptor β subunit (IRβ), insulin receptor substrates 1/2 (IRS1, IRS2) and hexokinase-II levels were determined by Western blot. Skeletal muscle insulin sensitivity was assessed by in vivo insulin-induced Akt phosphorylation (Western blot). Food intake and hypothalamic NPY mRNA expression (by qPCR) were also evaluated. Fasted insulin and HOMA-IR were augmented in GABAB1KO males, with no alterations in females. Areas under the curve (AUC) for GTT and ITT were increased in GABAB1KO mice of both genders, indicating compromised insulin sensitivity. No genotype differences were observed in IST, GST or in IRβ, IRS1, IRS2 and hexokinase-II expression. Akt activation was severely impaired in GABAB1KO males while no alterations were observed in females. GABAB1KO mice showed increased food intake and NPY expression. Glucose metabolism and energy balance disruptions were more pronounced in GABAB1KO males, which develop peripheral insulin resistance probably due to augmented insulin secretion. Metabolic alterations in females were milder and possibly due to previously described reproductive disorders, such as persistent estrus. Copyright © 2012 Elsevier Inc. All rights reserved.

Cardiac and vascular changes in elderly atherosclerotic mice: the influence of gender

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Pereira Thiago MC

2010-08-01

Full Text Available Abstract Background Although advanced age is considered a risk factor for several diseases, the impact of gender on age-associated cardiovascular diseases, such as atherosclerotic processes and valvular diseases, remains not completely clarified. The present study was designed to assess aortic valve morphology and function and vascular damage in elderly using the apolipoprotein E knockout (ApoE KO mouse. Our hypothesis was that advanced age-related cardiovascular changes are aggravated in atherosclerotic male mice. Methods The grade (0 to 4 of aortic regurgitation was evaluated through angiography. In addition, vascular lipid deposition and senescence were evaluated through histochemical analyses in aged male and female ApoE KO mice, and the results were compared to wild-type C57BL/6J (C57 mice. Results Aortic regurgitation was observed in 92% of the male ApoE KO mice and 100% of the male C57 mice. Comparatively, in age-matched female ApoE KO and C57 mice, aortic regurgitation was observed in a proportion of 58% and 53%, respectively. Histological analysis of the aorta showed an outward (positive remodeling in ApoE KO mice (female: 1.86 ± 0.15; male: 1.89 ± 0.68 using C57 groups as reference values. Histochemical evaluation of the aorta showed lipid deposition and vascular senescence only in the ApoE KO group, which were more pronounced in male mice. Conclusion The data show that male gender contributes to the progression of aortic regurgitation and that hypercholesterolemia and male gender additively contribute to the occurrence of lipid deposition and vascular senescence in elderly mice.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

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Zhang, Yao; Cheng, Yajun; Hansen, Gert H

2011-01-01

) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly....... The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof...... for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function....

Lacking Ketohexokinase-A Exacerbates Renal Injury in Streptozotocin-induced Diabetic Mice.

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Doke, Tomohito; Ishimoto, Takuji; Hayasaki, Takahiro; Ikeda, Satsuki; Hasebe, Masako; Hirayama, Akiyoshi; Soga, Tomoyoshi; Kato, Noritoshi; Kosugi, Tomoki; Tsuboi, Naotake; Lanaspa, Miguel A; Johnson, Richard J; Kadomatsu, Kenji; Maruyama, Shoichi

2018-03-28

Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6 J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediates levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD + level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD. Copyright © 2018. Published by Elsevier Inc.

Fluoxetine protection in decompression sickness in mice is enhanced by blocking TREK-1 potassium channel with the spadin antidepressant.

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Nicolas eVallée

2016-02-01

Full Text Available In mice, disseminated coagulation, inflammation and ischemia induce neurological damages that can lead to the death. These symptoms result from circulating bubbles generated by a pathogenic decompression. An acute fluoxetine treatment or the presence of the TREK-1 potassium channel increased the survival rate when mice are subjected to an experimental dive/decompression protocol. This is a paradox because fluoxetine is a blocker of TREK-1 channels. First, we studied the effects of an acute dose of fluoxetine (50mg/kg in wild-type (WT and TREK-1 deficient mice (Knockout homozygous KO and heterozygous HET. Then, we combined the same fluoxetine treatment with a five-day treatment by spadin, in order to specifically block TREK-1 activity (KO-like mice. KO and KO-like mice could be regarded as antidepressed models.167 mice (45 WTcont 46 WTflux 30 HETflux and 46 KOflux constituting the flux-pool and 113 supplementary mice (27 KO-like 24 WTflux2 24 KO-likeflux 21 WTcont2 17 WTno dive constituting the spad-pool were included in this study. Only 7% of KO-TREK-1 treated with fluoxetine (KOflux and 4% of mice treated with both spadin and fluoxetine (KO-likeflux died from decompression sickness (DCS symptoms. These values are much lower than those of WT control (62% or KO-like mice (41%. After the decompression protocol, mice showed a significant consumption of their circulating platelets and leukocytes.Spadin antidepressed mice were more likely to declare DCS. Nevertheless, which had both blocked TREK-1 channel and were treated with fluoxetine were better protected against DCS. We conclude that the protective effect of such an acute dose of fluoxetine is enhanced when TREK-1 is inhibited. We confirmed that antidepressed models may have worse DCS outcomes, but a concomitant fluoxetine treatment not only decreases DCS severity but increases the survival rate.

Mouse arsenic (+3 oxidation state) methyltransferase genotype affects metabolism and tissue dosimetry of arsenicals after arsenite administration in drinking water.

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Chen, Baowei; Arnold, Lora L; Cohen, Samuel M; Thomas, David J; Le, X Chris

2011-12-01

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes methylation of inorganic arsenic (iAs) producing a number of methylated arsenic metabolites. Although methylation has been commonly considered a pathway for detoxification of arsenic, some highly reactive methylated arsenicals may contribute to toxicity associated with exposure to inorganic arsenic. Here, adult female wild-type (WT) C57BL/6 mice and female As3mt knockout (KO) mice received drinking water that contained 1, 10, or 25 ppm (mg/l) of arsenite for 33 days and blood, liver, kidney, and lung were taken for arsenic speciation. Genotype markedly affected concentrations of arsenicals in tissues. Summed concentrations of arsenicals in plasma were higher in WT than in KO mice; in red blood cells, summed concentrations of arsenicals were higher in KO than in WT mice. In liver, kidney, and lung, summed concentrations of arsenicals were greater in KO than in WT mice. Although capacity for arsenic methylation is much reduced in KO mice, some mono-, di-, and tri-methylated arsenicals were found in tissues of KO mice, likely reflecting the activity of other tissue methyltransferases or preabsorptive metabolism by the microbiota of the gastrointestinal tract. These results show that the genotype for arsenic methylation determines the phenotypes of arsenic retention and distribution and affects the dose- and organ-dependent toxicity associated with exposure to inorganic arsenic.

Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice.

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Reham Khalaf-Nazzal

Full Text Available Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.

The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

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Bin Qiu

2016-08-01

Full Text Available FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1 Fkbp5 KO and wild-type (WT EtOH consumption was tested using a two-bottle choice paradigm; (2 The EtOH elimination rate was measured after intraperitoneal (IP injection of 2.0 g/kg EtOH; (3 Blood alcohol concentration (BAC was measured after 3 h limited access of alcohol; (4 Brain region expression of Fkbp5 was identified using LacZ staining; (5 Baseline corticosterone (CORT was assessed. Additionally, two SNPs, rs1360780 (C/T and rs3800373 (T/G, were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162 from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT. Finally, single nucleotide polymorphisms (SNPs in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

Long-lasting Effects of Minocycline on Behavior in Young but not Adult Fragile X Mice

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Dansie, Lorraine E.; Phommahaxay, Kelly; Okusanya, Ayodeji G.; Uwadia, Jessica; Huang, Mike; Rotschafer, Sarah E.; Razak, Khaleel A.; Ethell, Douglas W.; Ethell, Iryna M.

2013-01-01

Fragile X Syndrome (FXS) is the most common single-gene inherited form of intellectual disability with behaviors characteristic of autism. People with FXS display childhood seizures, hyperactivity, anxiety, developmental delay, attention deficits, and visual-spatial memory impairment, as well as a propensity for obsessive-compulsive disorder (OCD). Several of these aberrant behaviors and FXS-associated synaptic irregularities also occur in “fragile X mental retardation gene” knock-out (Fmr1 KO) mice. We previously reported that minocycline promotes the maturation of dendritic spines - postsynaptic sites for excitatory synapses - in the developing hippocampus of Fmr1 KO mice, which may underlie the beneficial effects of minocycline on anxiolytic behavior in young Fmr1 KO mice. In this study, we compared the effectiveness of minocycline treatment in young and adult Fmr1 KO mice, and determined the dependence of behavioral improvements on short-term versus long-term minocycline administration. We found that 4 and 8 week long treatments significantly reduced locomotor activity in both young and adult Fmr1 KO mice. Some behavioral improvements persisted in young mice post-treatment, but in adults the beneficial effects were lost soon after minocycline treatment was stopped. We also show, for the first time, that minocycline treatment partially attenuates the number and severity of audiogenic seizures in Fmr1 KO mice. This report provides further evidence that minocycline treatment has immediate and long-lasting benefits on FXS-associated behaviors in the Fmr1 KO mouse model. PMID:23660195

Effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and on atherogenesis in apolipoprotein E knock-out mice

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Portugal L.R.

2006-01-01

Full Text Available Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E knock-out (KO mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

Selegiline Ameliorates Depression-Like Behavior in Mice Lacking the CD157/BST1 Gene, a Risk Factor for Parkinson’s Disease

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Satoka Kasai

2017-05-01

Full Text Available Parkinson’s disease (PD, a neurodegenerative disorder, is accompanied by various non-motor symptoms including depression and anxiety, which may precede the onset of motor symptoms. Selegiline is an irreversible monoamine oxidase-B (MAO-B inhibitor, and is widely used in the treatment of PD and major depression. However, there are few reports about the effects of selegiline on non-motor symptoms in PD. The aim of this study was to explore the antidepressant and anxiolytic effects of selegiline, using CD157/BST1 knockout (CD157 KO mouse, a PD-related genetic model displaying depression and anxiety, compared with other antiparkinsonian drugs and an antidepressant, and was to investigate the effects of selegiline on biochemical parameters in emotion-related brain regions. A single administration of selegiline (1–10 mg/kg dose-dependently reduced immobility time in the forced swimming test (FST in CD157 KO mice, but not C57BL/6N wild-type (WT mice. At 10 mg/kg, but not 3 mg/kg, selegiline significantly increased climbing time in CD157 KO mice. A single administration of the antiparkinsonian drugs pramipexole (a dopamine (DA D2/D3 receptor agonist or rasagiline (another MAO-B inhibitor, and repeated injections of a noradrenergic and specific serotonergic antidepressant (NaSSA, mirtazapine, also decreased immobility time, but did not increase climbing time, in CD157 KO mice. The antidepressant-like effects of 10 mg/kg selegiline were comparable to those of 10 mg/kg rasagiline, and tended to be stronger than those of 1 mg/kg rasagiline. After the FST, CD157 KO mice showed decreases in striatal and hippocampal serotonin (5-HT content, cortical norepinephrine (NE content, and plasma corticosterone concentration. A single administration of selegiline at 10 mg/kg returned striatal 5-HT, cortical NE, and plasma corticosterone levels to those observed in WT mice. In the open field test (OFT, repeated administration of mirtazapine had anxiolytic effects

Long-Term Effectiveness of the Zilver PTX Drug-Eluting Stent for Femoropopliteal Peripheral Artery Disease in Patients with No Patent Tibial Runoff Vessels-Results from the Zilver PTX Japan Post-Market Surveillance Study.

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Cipollari, Stefano; Yokoi, Hiroyoshi; Ohki, Takao; Kichikawa, Kimihiko; Nakamura, Masato; Komori, Kimihiro; Nanto, Shinsuke; O'Leary, Erin E; Lottes, Aaron E; Saunders, Alan T; Dake, Michael D

2018-01-01

To evaluate 2-year results of the Zilver PTX (Cook Medical, Bloomington, Indiana) drug-eluting stent (DES) for femoropopliteal peripheral artery disease (PAD) in patients with no continuous patent infrapopliteal runoff arteries compared with patients with ≥ 1 continuous patent runoff vessels. A retrospective analysis of patients with femoropopliteal PAD enrolled in the Zilver PTX Post-Market Surveillance Study in Japan was performed. There were no exclusion criteria. Outcomes, including freedom from target lesion revascularization (TLR), patency, and clinical benefit, for the no-runoff group (n = 54) were compared with the runoff group (n = 846). The 2 groups were similar in terms of demographics, lesion characteristics, and comorbidities (P > .05). There was a higher incidence of critical limb ischemia in the no-runoff group compared with the runoff group (44.8% vs 19.7%; P < .01). There were 3 amputations (5.6%) in the no-runoff group versus 7 amputations (0.8%) in the runoff group (P = .02). At 2 years, freedom from TLR rates were 81.3% versus 83.8% (P = .87), patency rates were 68.4% versus 70.7% (P = .95), and clinical benefit rates were 73.7% versus 80.0% (P = .16) in the no-runoff versus runoff group, respectively. Results in patients with no continuous patent tibial runoff were favorable through 2 years and similar to results for patients with ≥ 1 continuous patent runoff vessels, indicating that the Zilver PTX DES may be a valid treatment option for patients with these difficult-to-treat lesions. Copyright © 2017 SIR. Published by Elsevier Inc. All rights reserved.

Effects of Eaf2 gene knockout on cataract induced by ultraviolet irradiation in mice

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Yan-Hua Jiang

2016-02-01

Full Text Available AIM:To evaluate the effects of Eaf2 gene knockout on cataract in mice induced by ultraviolet irradiation.METHODS:Fifteen wild type mice were used as the control group, and 10 Eaf2 KO mice were used as the experimental group. The 14-week mice were taken as the research objects in the two groups. So the subgroups were: WT -nonUV, WT -UV, Eaf2 KO-nonUV and Eaf2 KO-UV, a total of 4 groups. Observe the lens of mice in vivo with slit lamp microscope, grade the lens opacity with Lens Opacities Classification System II(LOCSII. Then the mice were sacrificed by breaking the neck, the lens were removed and were observed by dark field microscopy. According to the captured images, the proportion of cataract region was analyzed using Image J software. The data of the two groups were statistically analyzed.RESULTS: The results detected by the two methods were similar. In WT-UV group and Eaf2 KO-UV group, the degree of lens opacity was significantly higher than those of WT-nonUV group and Eaf2 KO-nonUV group. The lens opacity of WT-UV group was significantly higher than that in Eaf2 KO-UV group, and the difference was statistically significant(PCONCLUSION: Ultraviolet radiation can lead to the formation of cataract in mice. Eaf2 protein can promote the formation of cataract in mice caused by ultraviolet.

Mice lacking glutamate carboxypeptidase II develop normally, but are less susceptible to traumatic brain injury.

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Gao, Yang; Xu, Siyi; Cui, Zhenwen; Zhang, Mingkun; Lin, Yingying; Cai, Lei; Wang, Zhugang; Luo, Xingguang; Zheng, Yan; Wang, Yong; Luo, Qizhong; Jiang, Jiyao; Neale, Joseph H; Zhong, Chunlong

2015-07-01

Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia-bound GCPII mediates the hydrolysis of the neurotransmitter N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity associated with enhanced glutamate transmission under pathological conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3-5 of GCPII. This generated a new GCPII KO mice line with no overt differences in standard neurological behavior compared to their wild-type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addition, GCPII gene KO reduced TBI-induced deficits in long-term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathological protection with improved long-term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors. © 2015 International Society for Neurochemistry.

Distinct motor impairments of dopamine D1 and D2 receptor knockout mice revealed by three types of motor behavior

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Toru eNakamura

2014-07-01

Full Text Available Both D1R and D2R knock out (KO mice of the major dopamine receptors show significant motor impairments. However, there are some discrepant reports, which may be due to the differences in genetic background and experimental procedures. In addition, only few studies directly compared the motor performance of D1R and D2R KO mice. In this paper, we examined the behavioral difference among N10 congenic D1R and D2R KO, and wild type (WT mice. First, we examined spontaneous motor activity in the home cage environment for consecutive five days. Second, we examined motor performance using the rota-rod task, a standard motor task in rodents. Third, we examined motor ability with the Step-Wheel task in which mice were trained to run in a motor-driven turning wheel adjusting their steps on foothold pegs to drink water. The results showed clear differences among the mice of three genotypes in three different types of behavior. In monitoring spontaneous motor activities, D1R and D2R KO mice showed higher and lower 24 h activities, respectively, than WT mice. In the rota-rod tasks, at a low speed, D1R KO mice showed poor performance but later improved, whereas D2R KO mice showed a good performance at early days without further improvement. When first subjected to a high speed task, the D2R KO mice showed poorer rota-rod performance at a low speed than the D1R KO mice. In the Step-Wheel task, across daily sessions, D2R KO mice increased the duration that mice run sufficiently close to the spout to drink water, and decreased time to touch the floor due to missing the peg steps and number of times the wheel was stopped, which performance was much better than that of D1R KO mice. These incongruent results between the two tasks for D1R and D2R KO mice may be due to the differences in the motivation for the rota-rod and Step-Wheel tasks, aversion- and reward-driven, respectively. The Step-Wheel system may become a useful tool for assessing the motor ability of WT

Genetic Deletion and Pharmacological Inhibition of PI3Kγ Reduces Neutrophilic Airway Inflammation and Lung Damage in Mice with Cystic Fibrosis-Like Lung Disease

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Maria Galluzzo

2015-01-01

Full Text Available Purpose. Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF. Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kγ plays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kγ on airway inflammation and structural lung damage in a mouse model of CF lung disease. Methods. βENaC overexpressing mice (βENaC-Tg were backcrossed with PI3Kγ-deficient (PI3KγKO mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF. Furthermore, we assessed the effect of a specific PI3Kγ inhibitor (AS-605240 on inflammatory cell number in BALF. Results. Genetic deletion of PI3Kγ decreased neutrophil numbers in BALF of PI3KγKO/βENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kγ inhibitor AS-605240 decreased the number of neutrophils in BALF of βENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme. Conclusions. These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kγ in reducing chronic neutrophilic inflammation in CF-like lung disease in vivo.

Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12-dimethylbenz[a]anthracene

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Becks, Lisa; Shi, Runhua; McLarty, Jerry; Pruitt, Kevin; Zhang, Songlin; Kleiner-Hancock, Heather E; Prince, Misty; Burson, Hannah; Christophe, Christopher; Broadway, Mason; Itoh, Ken; Yamamoto, Masayuki; Mathis, Michael; Orchard, Elysse

2010-01-01

Activation of nuclear factor erythroid 2-related factor (Nrf2), which belongs to the basic leucine zipper transcription factor family, is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1 and peroxiredoxin 1, by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE, and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis. Premalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics, and the Mann-Whitney U-test. All mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice, there was a dramatic increase in mammary carcinoma growth rate, size, and weight. Although there was no difference in overall survival, the KO mice had significantly lower mammary tumor-free survival. Also, in the KO mammary carcinomas, the active forms of NF-κB and β-catenin were increased ~2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed, including lymphomas. Interestingly, the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice. We report, for the first time, that there was no apparent difference in the formation of premalignant lesions, but rather, the KO mice exhibited rapid, aggressive mammary carcinoma progression

Inhibiting Heat-Shock Protein 90 Reverses Sensory Hypoalgesia in Diabetic Mice

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Michael J Urban

2010-07-01

Full Text Available Increasing the expression of Hsp70 (heat-shock protein 70 can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R, 3R, 4S, 5R-3, 4-dihydroxy-5-methoxy-6, 6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

Ethanol-related behaviors in mice lacking the sigma-1 receptor.

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Valenza, Marta; DiLeo, Alyssa; Steardo, Luca; Cottone, Pietro; Sabino, Valentina

2016-01-15

The Sigma-1 receptor (Sig-1R) is a chaperone protein that has been implicated in drug abuse and addiction. Multiple studies have characterized the role the Sig-1R plays in psychostimulant addiction; however, fewer studies have specifically investigated its role in alcohol addiction. We have previously shown that antagonism of the Sig-1R reduces excessive drinking and motivation to drink, whereas agonism induces binge-like drinking in rodents. The objectives of these studies were to investigate the impact of Sig-1R gene deletion in C57Bl/6J mice on ethanol drinking and other ethanol-related behaviors. We used an extensive panel of behavioral tests to examine ethanol actions in male, adult mice lacking Oprs1, the gene encoding the Sig-1R. To compare ethanol drinking behavior, Sig-1 knockout (KO) and wild type (WT) mice were subject to a two-bottle choice, continuous access paradigm with different concentrations of ethanol (3-20% v/v) vs. water. Consumption of sweet and bitter solutions was also assessed in Sig-1R KO and WT mice. Finally, motor stimulant sensitivity, taste aversion and ataxic effects of ethanol were assessed. Sig-1R KO mice displayed higher ethanol intake compared to WT mice; the two genotypes did not differ in their sweet or bitter taste perception. Sig-1R KO mice showed lower sensitivity to ethanol stimulant effects, but greater sensitivity to its taste aversive effects. Ethanol-induced sedation was instead unaltered in the mutants. Our results prove that the deletion of the Sig-1R increases ethanol consumption, likely by decreasing its rewarding effects, and therefore indicating that the Sig-1R is involved in modulation of the reinforcing effects of alcohol. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

2017-11-01

Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency. Copyright © 2017 Endocrine Society.

Acute heat-evoked temperature sensation is impaired but not abolished in mice lacking TRPV1 and TRPV3 channels.

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Marics, Irène; Malapert, Pascale; Reynders, Ana; Gaillard, Stéphane; Moqrich, Aziz

2014-01-01

The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.

Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

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Irene Cuadrado

Full Text Available Inhibition of Extracellular Matrix degradation by nitric oxide (NO induces cardiac protection against coronary ischemia/reperfusion (IR. Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN stimulates enzymatic activation of matrix metalloproteinases (MMPs in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2 knockout (KO mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9, in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF. NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6. The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5, or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

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Cuadrado, Irene; Castejon, Borja; Martin, Ana M; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis; Zaragoza, Carlos

2016-01-01

Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

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Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

2014-01-01

Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor flox/flox mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function

Attenuated lung fibrosis in interleukin 6 knock-out mice after C-ion irradiation to lung

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Saito-Fujita, Tomoko; Iwakawa, Mayumi; Nakamura, Etsuko; Nakawatari, Miyako; Fujita, Hidetoshi; Moritake, Takashi; Imai, Takashi

2011-01-01

There is a great deal of evidence that a cyclic cascade of inflammatory cytokines, together with the activation of macrophages, is initiated very early after irradiation to develop lung fibrosis in a late phase. To understand the persistent effects of cytokines, the cytokine gene of knock out or transgenic mouse is one of the useful tools. In this study, we evaluated a role of a key molecule, interleukin-6 (IL-6), in the late-phase inflammatory response and subsequent fibrotic changes after irradiation using wild-type (WT) and IL-6 knock out (IL-6 KO) mice. The mice underwent thoracic irradiation with 10 Gy of C-ion beam or sham-irradiation and were examined by histology. Immunoreactivity for IL-6 was induced at the site of bronchiolar epithelium, in pneumocytes and in monocytes by C-ion irradiation. At 24 weeks after irradiation, the infiltration of macrophages, detected by positive immunohistological staining with Mac3 antibody, was observed in alveolar spaces both in WT and IL-6 KO mice. The thickening of bronchiolar and alveolar walls exhibited in WT mice, but not KO mice, and fibrotic changes detected by Masson-Trichrome staining, were observed only in the lungs of WT mice, while it was attenuated in IL-6 KO mice. These results indicated that IL-6 might not be essential for activating macrophages in the late phase, but plays an important role for fibrotic changes of the alveolar wall after irradiation. (author)

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

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Holgersen, Kristine; Dobie, Ross; Farquharson, Colin; vanʼt Hof, Rob; Ahmed, Syed Faisal; Hansen, Axel Kornerup; Holm, Thomas L

2015-02-01

Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut inflammation in an experimental colitis model. Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia and focal transmural inflammation, which was consistent with Crohn's disease-like pathology. The gut inflammation was accompanied by a 14% and 12% reduction in trabecular thickness relative to piroxicam-treated wild type and untreated wild type mice, respectively (P < 0.001). The trabecular bone structure was also changed in PAC IL-10 k.o. mice, whereas no differences in cortical bone geometry were observed. The trabecular thickness was inversely correlated with serum levels of CTX (r = -0.93, P = 0.006). Moreover, numerous inflammatory mediators, including RANKL and osteoprotegerin, were significantly increased in the colon of PAC IL-10 k.o. mice. PAC IL-10 k.o. mice develop bone loss and changed trabecular structure, as a result of increased bone resorption. Thus, the PAC IL-10 k.o. model could be a useful experimental model in preclinical research of inflammatory bowel disease-associated bone loss.

Dual AAV/IL-10 Plus STAT3 Anti-Inflammatory Gene Delivery Lowers Atherosclerosis in LDLR KO Mice, but without Increased Benefit

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Maohua Cao

2012-01-01

Full Text Available Both IL-10 and STAT3 are in the same signal transduction pathway, with IL-10-bound IL10 receptor (R acting through STAT3 for anti-inflammatory effect. To investigate possible therapeutic synergism, we delivered both full-length wild-type human (h STAT3 and hIL-10 genes by separate adenoassociated virus type 8 (AAV8 tail vein injection into LDLR KO on HCD. Compared to control Neo gene-treated animals, individual hSTAT3 and hIL-10 delivery resulted in significant reduction in atherogenesis, as determined by larger aortic lumen size, thinner aortic wall thickness, and lower blood velocity (all statistically significant. However, dual hSTAT3/hIL-10 delivery offered no improvement in therapeutic effect. Plasma cholesterol levels in dual hSTAT3/hIL-10-treated animals were statistically higher compared to hIL-10 alone. While no advantage was seen in this case, we consider that the dual gene approach has intrinsic merit, but properly chosen partnered genes must be used.

Pregnenolone rescues schizophrenia-like behavior in dopamine transporter knockout mice.

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Peiyan Wong

Full Text Available Pregnenolone belongs to a class of endogenous neurosteroids in the central nervous system (CNS, which has been suggested to enhance cognitive functions through GABA(A receptor signaling by its metabolites. It has been shown that the level of pregnenolone is altered in certain brain areas of schizophrenic patients, and clozapine enhances pregnenolone in the CNS in rats, suggesting that pregnenolone could be used to treat certain symptoms of schizophrenia. In addition, early phase proof-of-concept clinical trials have indicated that pregnenolone is effective in reducing the negative symptoms and cognitive deficits of schizophrenia patients. Here, we evaluate the actions of pregnenolone on a mouse model for schizophrenia, the dopamine transporter knockout mouse (DAT KO. DAT KO mice mirror certain symptoms evident in patients with schizophrenia, such as the psychomotor agitation, stereotypy, deficits of prepulse inhibition and cognitive impairments. Following acute treatment, pregnenolone was found to reduce the hyperlocomotion, stereotypic bouts and pre-pulse inhibition (PPI deficits in DAT KO mice in a dose-dependent manner. At 60 mg/kg of pregnenolone, there were no significant differences in locomotor activities and stereotypy between wild-type and DAT KO mice. Similarly, acute treatment of 60 mg/kg of pregnenolone fully rescued PPI deficits of DAT KO mice. Following chronic treatment with pregnenolone at 60 mg/kg, the cognitive deficits of DAT KO mice were rescued in the paradigms of novel object recognition test and social transmission of food preference test. Pregnenolone thus holds promise as a therapeutic candidate in schizophrenia.

Altered social behaviours in neurexin 1α knockout mice resemble core symptoms in neurodevelopmental disorders.

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Hannah Mary Grayton

Full Text Available BACKGROUND: Copy number variants have emerged as an important genomic cause of common, complex neurodevelopmental disorders. These usually change copy number of multiple genes, but deletions at 2p16.3, which have been associated with autism, schizophrenia and mental retardation, affect only the neurexin 1 gene, usually the alpha isoform. Previous analyses of neurexin 1α (Nrxn1α knockout (KO mouse as a model of these disorders have revealed impairments in synaptic transmission but failed to reveal defects in social behaviour, one of the core symptoms of autism. METHODS: We performed a detailed investigation of the behavioural effects of Nrxn1α deletion in mice bred onto a pure genetic background (C57BL/6J to gain a better understanding of its role in neurodevelopmental disorders. Wildtype, heterozygote and homozygote Nrxn1α KO male and female mice were tested in a battery of behavioural tests (n = 9-16 per genotype, per sex. RESULTS: In homozygous Nrxn1α KO mice, we observed altered social approach, reduced social investigation, and reduced locomotor activity in novel environments. In addition, male Nrxn1α KO mice demonstrated an increase in aggressive behaviours. CONCLUSIONS: These are the first experimental data that associate a deletion of Nrxn1α with alterations of social behaviour in mice. Since this represents one of the core symptom domains affected in autism spectrum disorders and schizophrenia in humans, our findings suggest that deletions within NRXN1 found in patients may be responsible for the impairments seen in social behaviours, and that the Nrxn1α KO mice are a useful model of human neurodevelopmental disorder.

Altered Social Behaviours in Neurexin 1α Knockout Mice Resemble Core Symptoms in Neurodevelopmental Disorders

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Grayton, Hannah Mary; Missler, Markus

2013-01-01

Background Copy number variants have emerged as an important genomic cause of common, complex neurodevelopmental disorders. These usually change copy number of multiple genes, but deletions at 2p16.3, which have been associated with autism, schizophrenia and mental retardation, affect only the neurexin 1 gene, usually the alpha isoform. Previous analyses of neurexin 1α (Nrxn1α) knockout (KO) mouse as a model of these disorders have revealed impairments in synaptic transmission but failed to reveal defects in social behaviour, one of the core symptoms of autism. Methods We performed a detailed investigation of the behavioural effects of Nrxn1α deletion in mice bred onto a pure genetic background (C57BL/6J) to gain a better understanding of its role in neurodevelopmental disorders. Wildtype, heterozygote and homozygote Nrxn1α KO male and female mice were tested in a battery of behavioural tests (n = 9–16 per genotype, per sex). Results In homozygous Nrxn1α KO mice, we observed altered social approach, reduced social investigation, and reduced locomotor activity in novel environments. In addition, male Nrxn1α KO mice demonstrated an increase in aggressive behaviours. Conclusions These are the first experimental data that associate a deletion of Nrxn1α with alterations of social behaviour in mice. Since this represents one of the core symptom domains affected in autism spectrum disorders and schizophrenia in humans, our findings suggest that deletions within NRXN1 found in patients may be responsible for the impairments seen in social behaviours, and that the Nrxn1α KO mice are a useful model of human neurodevelopmental disorder. PMID:23840597

Effects of gender on locomotor sensitivity to amphetamine, body weight, and fat mass in regulator of G protein signaling 9 (RGS9) knockout mice.

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Walker, Paul D; Jarosz, Patricia A; Bouhamdan, Mohamad; MacKenzie, Robert G

2015-01-01

Regulator of G-protein signaling (RGS) protein 9-2 is enriched in the striatum where it modulates dopamine and opioid receptor-mediated signaling. RGS9 knockout (KO) mice show increased psychostimulant-induced behavioral sensitization, as well as exhibit higher body weights and greater fat accumulation compared to wild-type (WT) littermates. In the present study, we found gender influences on each of these phenotypic characteristics. Female RGS9 KO mice exhibited greater locomotor sensitization to amphetamine (1.0mg/kg) treatment as compared to male RGS9 KO mice. Male RGS9 KO mice showed increased body weights as compared to male WT littermates, while no such differences were detected in female mice. Quantitative magnetic resonance showed that male RGS9 KO mice accumulated greater fat mass vs. WT littermates at 5months of age. Such observations could not be explained by increased caloric consumption since male and female RGS9 KO mice demonstrated equivalent daily food intake as compared to their respective WT littermates. Although indirect calorimetry methods found decreased oxygen consumption and carbon dioxide production during the 12-hour dark phase in male RGS9 KO vs. WT mice which are indicative of less energy expenditure, male RGS9 KO mice exhibited lower levels of locomotor activity during this period. Genotype had no effect on metabolic activities when KO and WT groups were compared under fasting vs. feeding treatments. In summary, these results highlight the importance of factoring gender into the experimental design since many studies conducted in RGS9 KO mice utilize locomotor activity as a measured outcome. Copyright © 2014. Published by Elsevier Inc.

Increased consumption of ethanol and sugar water in mice lacking the dopamine D2 long receptor.

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Bulwa, Zachary B; Sharlin, Jordan A; Clark, Peter J; Bhattacharya, Tushar K; Kilby, Chessa N; Wang, Yanyan; Rhodes, Justin S

2011-11-01

Individual differences in dopamine D2 receptor (D2R) expression in the brain are thought to influence motivation and reinforcement for ethanol and other rewards. D2R exists in two isoforms, D2 long (D2LR) and D2 short (D2SR), produced by alternative splicing of the same gene. The relative contributions of D2LR versus D2SR to ethanol and sugar water drinking are not known. Genetic engineering was used to produce a line of knockout (KO) mice that lack D2LR and consequently have increased expression of D2SR. KO and wild-type (WT) mice of both sexes were tested for intake of 20% ethanol, 10% sugar water and plain tap water using established drinking-in-the-dark procedures. Mice were also tested for effects of the D2 antagonist eticlopride on intake of ethanol to determine whether KO responses were caused by lack of D2LR or overrepresentation of D2SR. Locomotor activity on running wheels and in cages without wheels was also measured for comparison. D2L KO mice drank significantly more ethanol than WT in both sexes. KO mice drank more sugar water than WT in females but not in males. Eticlopride dose dependently decreased ethanol intake in all groups except male KO. KO mice were less physically active than WT in cages with or without running wheels. Results suggest that overrepresentation of D2SR contributes to increased intake of ethanol in the KO mice. Decreasing wheel running and general levels of physical activity in the KO mice rules out the possibility that higher intake results from higher motor activity. Results extend the literature implicating altered expression of D2R in risk for addiction by delineating the contribution of individual D2R isoforms. These findings suggest that D2LR and D2SR play differential roles in consumption of alcohol and sugar rewards. Copyright © 2011 Elsevier Inc. All rights reserved.

Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

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Hatazawa, Yukino; Ono, Yusuke; Hirose, Yuma; Kanai, Sayaka; Fujii, Nobuharu L; Machida, Shuichi; Nishino, Ichizo; Shimizu, Takahiko; Okano, Masaki; Kamei, Yasutomi; Ogawa, Yoshihiro

2018-03-01

DNA methylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNA methyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy. We made skeletal muscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KO mice. Diminished mRNA and protein expression of Dnmt3a were observed in skeletal muscles as well as in satellite cells, which are important for muscle regeneration, in Dnmt3a-KO mice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5) mRNA was markedly increased in Dnmt3a-KO mice. The DNA methylation level of the Gdf5 promoter was markedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated by DNA methylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5) mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation via DNA methylation. This mechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia.-Hatazawa, Y., Ono, Y., Hirose, Y., Kanai, S., Fujii, N. L., Machida, S., Nishino, I., Shimizu, T., Okano, M., Kamei, Y., Ogawa, Y. Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Bex1 knock out mice show altered skeletal muscle regeneration

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

2007-01-01

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 Ca 2+ /CaM may be involved in skeletal muscle regeneration

CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury.

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Mathes, Denise; Weirather, Johannes; Nordbeck, Peter; Arias-Loza, Anahi-Paula; Burkard, Matthias; Pachel, Christina; Kerkau, Thomas; Beyersdorf, Niklas; Frantz, Stefan; Hofmann, Ulrich

2016-12-01

The present study analyzed the effect of CD4 + Forkhead box protein 3 negative (Foxp3 - ) T-cells and Foxp3 + CD4 + T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4 + T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4 + T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4 + T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4 + T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4 + T-cells in CD4 KO mice increased the infarct size only when including the Foxp3 + CD25 + subset. Depletion of CD4 + Foxp3 + T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CD4 + Foxp3 + T-cells enhance myocardial ischemia-reperfusion injury. CD4 + T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

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Suzuki, Katsumasa; Fukui, Hirokazu; Kayahara, Takahisa; Sawada, Mitsutaka; Seno, Hiroshi; Hiai, Hiroshi; Kageyama, Ryoichiro; Okano, Hideyuki; Chiba, Tsutomu

2005-01-01

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

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Wayne A Cabral

2014-06-01

Full Text Available Cyclophilin B (CyPB, encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO mice are small, with reduced femoral areal bone mineral density (aBMD, bone volume per total volume (BV/TV and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11% collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1 activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

Science.gov (United States)

Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

2014-01-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

Male aromatase-knockout mice exhibit normal levels of activity, anxiety and "depressive-like" symptomatology.

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Dalla, C; Antoniou, K; Papadopoulou-Daifoti, Z; Balthazart, J; Bakker, J

2005-09-08

It is well known that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood. It was recently found that male aromatase knockout mice (ArKO) deficient in estradiol due to a mutation in the aromatase gene have general deficits in coital behavior and are sexually less motivated. We wondered whether these behavioral deficits of ArKO males could be related to changes in activity, exploration, anxiety and "depressive-like" symptomatology. ArKO and wild type (WT) males were subjected to open field (OF), elevated plus maze (EPM), and forced swim tests (FST), after being exposed or not to chronic mild stress (CMS). CMS was used to evaluate the impact of chronic stressful procedures and to unveil possible differences between genotypes. There was no effect of genotype on OF, EPM and FST behavioral parameters. WT and ArKO mice exposed to CMS or not exhibited the same behavioral profile during these three types of tests. However, all CMS-exposed mice (ArKO and WT) spent less time in the center of the EPM. Additionally, floating duration measured in the FST increased between two tests in both WT and ArKO mice, though that increase was less prominent in mice previously subjected to CMS than in controls. Therefore, both ArKO and WT males displayed the same behavior and had the same response to CMS however CMS exposure slightly modified the behavior displayed by mice of both genotypes in the FST and EPM paradigms. These results show that ArKO males display normal levels of activity, exploration, anxiety and "depressive-like" symptomatology and thus their deficits in sexual behavior are specific in nature and do not result indirectly from other behavioral changes.

High-pressure synthesis and structural, physical properties of CaIr1-xPtxO3 and CaIr1-xRhxO3

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Hirai, S.; Bromiley, G. D.; Klemme, S.; Irifune, T.; Ohfuji, H.; Attfield, P.; Nishiyama, N.

2010-12-01

in terms of materials science applications. To our knowledge, this will be the first report on structural, magnetic and charge-transport properties of B-site substituted solid solutions of post-perovskite oxides with 4d/5d transition metals. High-quality polycrystalline samples of CaIr1-xPtxO3 and CaIr1-xRhxO3 have been obtained at high pressures, and structural, magnetic and charge-transport properties of the compounds will be reported. ODF analysis reveals that solutions of CaIrO3, CaPtO3 and CaRhO3 exhibit similar grain growth features to the mother compound, although growth in [0 1 0] plays a more dominant role than the growth in [0 0 1] for the solid solutions. CaIrO3 is a characteristic hard magnet suitable for applications such as magnetic recording, with TN = 108K. A new phase of CaIr1-xPtxO3 synthesized at a high P/T condition has Raman modes which resemble those of CaIrO3 perovskite, suggesting this phase has a perovskite structure.The instability of the perovskite phase of CaIr1-xPtxO3 reveals why the post-perovskite to peovskite phase transition has not been observed for CaPtO3 unlike the case for CaIrO3, CaRhO3 and CaRuO3.

CB2 cannabinoid receptors modulate HIF-1α and TIM-3 expression in a hypoxia-ischemia mouse model.

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Kossatz, Elk; Maldonado, Rafael; Robledo, Patricia

2016-12-01

The role of CB2 cannabinoid receptors (CB 2 R) in global brain lesions induced by hypoxia-ischemia (HI) insult is still unresolved. The aim of this study was to evaluate the involvement of CB 2 R in the behavioural and biochemical underpinnings related to brain damage induced by HI in adult mice, and the mechanisms involved. CB 2 R knockout (KO) mice and wild-type littermates (WT) underwent permanent ligation of the left common carotid artery and hypoxia. Behavioural measurements in the rotarod, beam walking, object recognition, open field, and Irwin tests were carried out 24h, 72h and 7 days. In KO mice, more extensive brain injury was observed. Behavioural deficits in the Irwin test were observed in both genotypes; while WT mice showed progressive recovery by day 7, KO mice did not. Only KO mice showed alterations in motor learning, coordination and balance, and did not recover over time. A higher expression of microglia and astrocytes was observed in several brain areas of lesioned WT and KO mice. The possible alteration of the inflammatory-related factors HIF-1α and TIM-3 was evaluated in these animals. In both genotypes, HIF-1α and TIM-3 expression was observed in lesioned areas associated with activated microglia. However, the expression levels of these proteins were exacerbated in KO mice in several lesioned and non-lesioned brain structures. Our results indicate that CB 2 R may have a crucial neuroprotective role following HI insult through the modulation of the inflammatory-related HIF-1α/TIM-3 signalling pathway in microglia. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

An Examination of the Role of L-Glutamate and Inosine 5'-Monophosphate in Hedonic Taste-Guided Behavior by Mice Lacking the T1R1 + T1R3 Receptor.

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Blonde, Ginger D; Spector, Alan C

2017-06-01

The heterodimeric T1R1 + T1R3 receptor is considered critical for normal signaling of L-glutamate and 5'-ribonucleotides in the oral cavity. However, some taste-guided responsiveness remains in mice lacking one subunit of the receptor, suggesting that other receptors are sufficient to support some behaviors. Here, mice lacking both receptor subunits (KO) and wild-type (WT, both n = 13) mice were tested in a battery of behavioral tests. Mice were trained and tested in gustometers with a concentration series of Maltrin-580, a maltodextrin, in a brief-access test (10-s trials) as a positive control. Similar tests followed with monosodium glutamate (MSG) with and without the ribonucleotide inosine 5'-monophosphate (IMP), but always in the presence of the epithelial sodium channel blocker amiloride (A). Brief-access tests were repeated following short-term (30-min) and long-term (48-h) exposures to MSG + A + IMP and were also conducted with sodium gluconate replacing MSG. Finally, progressive ratio tests were conducted with Maltrin-580 or MSG + A + IMP, to assess appetitive behavior while minimizing satiation. Overall, MSG generated little concentration-dependent responding in either food-restricted WT or KO mice, even in combination with IMP. However, KO mice licked less to the amino acid stimuli, a measure of consummatory behavior in the brief-access tests. In contrast, both groups initiated a similar number of trials and had a similar breakpoint in the progressive ratio task, both measures of appetitive (approach) behavior. Collectively, these results suggest that while the T1R1 + T1R3 receptor is necessary for consummatory responding to MSG (+IMP), other receptors are sufficient to maintain appetitive responding to this "umami" stimulus complex in food-restricted mice. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

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Yamamoto, Hideko; Kamegaya, Etsuko; Hagino, Yoko; Takamatsu, Yukio; Sawada, Wakako; Matsuzawa, Maaya; Ide, Soichiro; Yamamoto, Toshifumi; Mishina, Masayoshi; Ikeda, Kazutaka

2017-01-01

The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT 2C ) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT 2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT 2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT 2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special

DBI/ACBP loss-of-function does not affect anxiety-like behaviour but reduces anxiolytic responses to diazepam in mice

DEFF Research Database (Denmark)

Budry, Lionel; Bouyakdan, Khalil; Tobin, Stephanie

2016-01-01

. Male and female ACBP(GFAP) KO and ACBP KO mice do not show significant changes in anxiety-like behaviour compared to control littermates during elevated plus maze (EPM) and open field (OF) tests. Surprisingly, ACBP(GFAP) KO and ACBP KO mice were unresponsive to the anxiolytic effect of a low dose...... of diazepam during EPM tests. In conclusion, our experiments using genetic ACBP loss-of-function models suggest that endozepines deficiency does not affect anxiety-like behaviour in mice and impairs the anxiolytic action of diazepam....

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

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Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Shan, Tizhong; Liang, Xinrong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

2014-09-12

Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

Podocyte-specific deletion of Rac1 leads to aggravation of renal injury in STZ-induced diabetic mice

International Nuclear Information System (INIS)

Ishizaka, Masanori; Gohda, Tomohito; Takagi, Miyuki; Omote, Keisuke; Sonoda, Yuji; Oliva Trejo, Juan Alejandro; Asao, Rin; Hidaka, Teruo; Asanuma, Katsuhiko; Horikoshi, Satoshi; Tomino, Yasuhiko

2015-01-01

Rac1, a GTPase of the Rho subfamily, has a crucial role in cytoskeletal architecture, as well as the regulation of cell migration and growth. However, renal injury in mice with podocyte-specific deletion of Rac1 has yet to be elucidated fully due to conflicting findings. Herein, we identified a possible role for Rac1 in podocytes of streptozotocin- (STZ) induced diabetic mice. The urinary albumin/creatinine ratio (ACR) in the knockout (KO) group was significantly higher than that in the wild type (WT) group at any week of age. A more marked ACR increase was observed in STZ/KO group than STZ/WT group, although ACR did increase with weeks of age in both diabetic groups. The kidney sections from diabetic mice revealed a glomerular hypertrophy with mesangial expansion, but there was no appreciable difference in glomerular findings under a light microscope between STZ/WT and STZ/KO mice. However, an electron microscopy analysis revealed that regardless of the presence or absence of diabetes, both KO (KO and STZ/KO) groups had a higher rate of foot process effacement compared with both WT (WT and STZ/WT) groups. The expression levels of the slit diaphragm protein, podocin, was reduced with the induction of diabetes, and the levels in the STZ/KO group experienced a further reduction compared with the STZ/WT group. The number of WT1-positive cells in the STZ/KO group was more significantly decreased than that in the other three groups. In contrast, the numbers of cleaved caspase 3- and TUNEL-positive cells in the glomeruli of the STZ/KO group were more increased than those in the STZ/WT group. Thus, this study provides evidence that podocyte-specific deletion of Rac1 results in morphological alteration in podocytes, and that the induction of apoptosis or decreased expression of the slit diaphragm proteins by hyperglycemic stimuli are associated with the progression of diabetic nephropathy.

Podocyte-specific deletion of Rac1 leads to aggravation of renal injury in STZ-induced diabetic mice

Energy Technology Data Exchange (ETDEWEB)

Ishizaka, Masanori [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Gohda, Tomohito, E-mail: goda@juntendo.ac.jp [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Takagi, Miyuki; Omote, Keisuke; Sonoda, Yuji [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Oliva Trejo, Juan Alejandro [Laboratory for Kidney Research (TMK Project), Medical Innovation Center, Kyoto University Graduate School of Medicine, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8397 (Japan); Asao, Rin; Hidaka, Teruo [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Asanuma, Katsuhiko [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Laboratory for Kidney Research (TMK Project), Medical Innovation Center, Kyoto University Graduate School of Medicine, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8397 (Japan); Horikoshi, Satoshi [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Tomino, Yasuhiko [Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Medical Corporation SHOWAKAI, 3-12-12 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023 (Japan)

2015-11-20

Rac1, a GTPase of the Rho subfamily, has a crucial role in cytoskeletal architecture, as well as the regulation of cell migration and growth. However, renal injury in mice with podocyte-specific deletion of Rac1 has yet to be elucidated fully due to conflicting findings. Herein, we identified a possible role for Rac1 in podocytes of streptozotocin- (STZ) induced diabetic mice. The urinary albumin/creatinine ratio (ACR) in the knockout (KO) group was significantly higher than that in the wild type (WT) group at any week of age. A more marked ACR increase was observed in STZ/KO group than STZ/WT group, although ACR did increase with weeks of age in both diabetic groups. The kidney sections from diabetic mice revealed a glomerular hypertrophy with mesangial expansion, but there was no appreciable difference in glomerular findings under a light microscope between STZ/WT and STZ/KO mice. However, an electron microscopy analysis revealed that regardless of the presence or absence of diabetes, both KO (KO and STZ/KO) groups had a higher rate of foot process effacement compared with both WT (WT and STZ/WT) groups. The expression levels of the slit diaphragm protein, podocin, was reduced with the induction of diabetes, and the levels in the STZ/KO group experienced a further reduction compared with the STZ/WT group. The number of WT1-positive cells in the STZ/KO group was more significantly decreased than that in the other three groups. In contrast, the numbers of cleaved caspase 3- and TUNEL-positive cells in the glomeruli of the STZ/KO group were more increased than those in the STZ/WT group. Thus, this study provides evidence that podocyte-specific deletion of Rac1 results in morphological alteration in podocytes, and that the induction of apoptosis or decreased expression of the slit diaphragm proteins by hyperglycemic stimuli are associated with the progression of diabetic nephropathy.

Increased susceptibility to diet-induced obesity in histamine-deficient mice

DEFF Research Database (Denmark)

Jørgensen, Emilie A; Vogelsang, Thomas W; Knigge, Ulrich

2006-01-01

in the development of high-fat diet (HFD)-induced obesity. METHODS: Histamine-deficient histidine decarboxylase knock-out (HDC-KO) mice and C57BL/6J wild-type (WT) mice were given either a standard diet (STD) or HFD for 8 weeks. Body weight, 24-hour caloric intake, epididymal adipose tissue size, plasma leptin...... weeks, whereas a significant difference in body weight gain was first observed after 5 weeks in WT mice. After 8 weeks 24-hour caloric intake was significantly lower in HFD- than in STD-fed WT mice. In HDC-KO mice no difference in caloric intake was observed between HFD- and STD-fed mice. After 8 weeks...

Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level

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Wang Jia

2009-11-01

Full Text Available Abstract Background Protein kinase C interacting protein (PKCI/HINT1 is a small protein belonging to the histidine triad (HIT family proteins. Its brain immunoreactivity is located in neurons and neuronal processes. PKCI/HINT1 gene knockout (KO mice display hyper-locomotion in response to D-amphetamine which is considered a positive symptom of schizophrenia in animal models. Postmortem studies identified PKCI/HINT1 as a candidate molecule for schizophrenia and bipolar disorder. We investigated the hypothesis that the PKCI/HINT1 gene may play an important role in regulating mood function in the CNS. We submitted PKCI/HINT1 KO mice and their wild type (WT littermates to behavioral tests used to study anti-depressant, anxiety like behaviors, and goal-oriented behavior. Additionally, as many mood disorders coincide with modifications of hypothalamic-pituitary-adrenal (HPA axis function, we assessed the HPA activity through measurement of plasma corticosterone levels. Results Compared to the WT controls, KO mice exhibited less immobility in the forced swim (FST and the tail suspension (TST tests. Activity in the TST tended to be attenuated by acute treatment with valproate at 300 mg/kg in KO mice. The PKCI/HINT1 KO mice presented less thigmotaxis in the Morris water maze and spent progressively more time in the lit compartment in the light/dark test. In a place navigation task, KO mice exhibited enhanced acquisition and retention. Furthermore, the afternoon basal plasma corticosterone level in PKCI/HINT1 KO mice was significantly higher than in the WT. Conclusion PKCI/HINT1 KO mice displayed a phenotype of behavioral and endocrine features which indicate changes of mood function, including anxiolytic-like and anti-depressant like behaviors, in conjunction with an elevated corticosterone level in plasma. These results suggest that the PKCI/HINT 1 gene could be important for the mood regulation function in the CNS.

7,8-Dihydroxyflavone Ameliorates Cognitive Impairment by Inhibiting Expression of Tau Pathology in ApoE-Knockout Mice

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Yang Tan

2016-11-01

Full Text Available 7,8-Dihydroxyflavone (7,8-DHF, a tyrosine kinase B (TrkB agonist that mimics the neuroprotective properties of brain-derived neurotrophic factor, which can not efficiently deliver into the brain, has been reported to be useful in ameliorating cognitive impairment in many diseases. Researches have indicated that apolipoprotein E-knockout (ApoE-KO mouse was associated with cognitive alteration via various mechanisms. Our present study investigated the possible mechanisms of cognitive impairment of ApoE-KO mouse fed with western type diet and the protective effects of 7,8-DHF in improving spatial learning and memory in ApoE-KO mouse. 5-weeks-old ApoE-KO mice and C57BL/6 mice were chronically treated with 7,8-DHF (with a dosage of 5mg/kg or vehicles orally for 25 weeks, and then subjected to Morris water maze at the age of 30 weeks to evaluate the cognitive performances. Afterwards, histology analysis and western blotting were performed. Spatial learning and memory deficits were observed in ApoE-KO mice, which were consistent with higher expression of active-asparaginyl endopeptidase (active-AEP as well as AEP-derived truncated tauN368 compared with normal group. In addition to that, long-term treatment of 7,8-DHF dramatically ameliorated cognitive decline in ApoE-KO mice, accompanied by the activation in phosphorylated protein kinase B (Akt/glycogen synthase kinase-3β (GSK-3β pathway and down-regulated expression of tau S396 and PHF-tau (phosphorylated tau at ser396 and ser404 epitope. These findings suggested that cognitive impairment of ApoE-KO mouse might associate with tau pathology and 7,8-DHF could activate AKT and then phosphorylate its downstream molecule to inhibit expression of abnormal tau, meanwhile, 7,8-DHF could reduce the expression of active-AEP and then inhibit production of truncated tauN368.

GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior

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Fuka Aizawa

2016-12-01

Full Text Available The free fatty acid receptor 1 (GPR40/FFAR1 is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO mice. The emotional behavior in wild and KO male mice was evaluated at 9–10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC–MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain.

Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

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Yokoi, Fumiaki; Chen, Huan-Xin; Dang, Mai Tu; Cheetham, Chad C; Campbell, Susan L; Roper, Steven N; Sweatt, J David; Li, Yuqing

2015-01-01

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA(ΔE)). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA(ΔE) does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.

Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

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Fumiaki Yokoi

Full Text Available DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A, which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG corresponding to a glutamic acid in the C-terminal region (torsinA(ΔE. Dyt1 ΔGAG heterozygous knock-in (KI mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs and normal theta-burst-induced long-term potentiation (LTP in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA(ΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.

Eicosapentaenoic acid ameliorates non-alcoholic steatohepatitis in a novel mouse model using melanocortin 4 receptor-deficient mice.

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Konuma, Kuniha; Itoh, Michiko; Suganami, Takayoshi; Kanai, Sayaka; Nakagawa, Nobutaka; Sakai, Takeru; Kawano, Hiroyuki; Hara, Mitsuko; Kojima, Soichi; Izumi, Yuichi; Ogawa, Yoshihiro

2015-01-01

Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH), while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS), where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA), a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.

Eicosapentaenoic acid ameliorates non-alcoholic steatohepatitis in a novel mouse model using melanocortin 4 receptor-deficient mice.

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Kuniha Konuma

Full Text Available Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH, while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS, where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA, a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.

Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

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Matthew DeVries

2010-03-01

Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

Role of METTL20 in regulating β-oxidation and heat production in mice under fasting or ketogenic conditions.

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Shimazu, Tadahiro; Furuse, Tamio; Balan, Shabeesh; Yamada, Ikuko; Okuno, Shuzo; Iwanari, Hiroko; Suzuki, Takehiro; Hamakubo, Takao; Dohmae, Naoshi; Yoshikawa, Takeo; Wakana, Shigeharu; Shinkai, Yoichi

2018-01-19

METTL20 is a seven-β-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) β subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher β-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when β-oxidation is highly activated.

Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice.

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Glendinning, John I; Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F; Vasselli, Joseph R; Sclafani, Anthony

2015-09-01

Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. Copyright © 2015 the American Physiological Society.

Rac1 in muscle is dispensable for improved insulin action after exercise in mice

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Sylow, Lykke; Møller, Lisbeth Liliendal Valbjørn; D'Hulst, Gommaar

2016-01-01

sensitivity in inducible muscle-specific Rac1 knockout (mKO) and wildtype littermate (WT) mice. Prior exercise enhanced whole body insulin sensitivity by 40% in WT mice and rescued the insulin intolerance in Rac1 mKO mice by improving whole body insulin sensitivity by 230%. In agreement, prior exercise...... significantly improved insulin sensitivity by 20% in WT and by 40% in Rac1 mKO soleus muscles. These findings suggest that muscle Rac1 is dispensable for the insulin sensitizing effect of exercise. Moreover, insulin resistance in Rac1 mKO mice can be completely normalized by prior exercise explaining why......Exercise has a potent insulin-sensitivity enhancing effect on skeletal muscle but the intracellular mechanisms that mediate this effect are not well understood. In muscle, Rac1 regulates both insulin- and contraction-stimulated glucose transport and is dysregulated in insulin resistant muscle...

Environmental Enrichment Ameliorates Behavioral Impairments Modeling Schizophrenia in Mice Lacking Metabotropic Glutamate Receptor 5.

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Burrows, Emma L; McOmish, Caitlin E; Buret, Laetitia S; Van den Buuse, Maarten; Hannan, Anthony J

2015-07-01

Schizophrenia arises from a complex interplay between genetic and environmental factors. Abnormalities in glutamatergic signaling have been proposed to underlie the emergence of symptoms, in light of various lines of evidence, including the psychotomimetic effects of NMDA receptor antagonists. Metabotropic glutamate receptor 5 (mGlu5) has also been implicated in the disorder, and has been shown to physically interact with NMDA receptors. To clarify the role of mGlu5-dependent behavioral expression by environmental factors, we assessed mGlu5 knockout (KO) mice after exposure to environmental enrichment (EE) or reared under standard conditions. The mGlu5 KO mice showed reduced prepulse inhibition (PPI), long-term memory deficits, and spontaneous locomotor hyperactivity, which were all attenuated by EE. Examining the cellular impact of genetic and environmental manipulation, we show that EE significantly increased pyramidal cell dendritic branching and BDNF protein levels in the hippocampus of wild-type mice; however, mGlu5 KO mice were resistant to these alterations, suggesting that mGlu5 is critical to these responses. A selective effect of EE on the behavioral response to the NMDA receptor antagonist MK-801 in mGlu5 KO mice was seen. MK-801-induced hyperlocomotion was further potentiated in enriched mGlu5 KO mice and treatment with MK-801 reinstated PPI disruption in EE mGlu5 KO mice only, a response that is absent under standard housing conditions. Together, these results demonstrate an important role for mGlu5 in environmental modulation of schizophrenia-related behavioral impairments. Furthermore, this role of the mGlu5 receptor is mediated by interaction with NMDA receptor function, which may inform development of novel therapeutics.

Abnormal social behavior, hyperactivity, impaired remote spatial memory, and increased D1-mediated dopaminergic signaling in neuronal nitric oxide synthase knockout mice

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Tanda Koichi

2009-06-01

Full Text Available Abstract Background Neuronal nitric oxide synthase (nNOS is involved in the regulation of a diverse population of intracellular messenger systems in the brain. In humans, abnormal NOS/nitric oxide metabolism is suggested to contribute to the pathogenesis and pathophysiology of some neuropsychiatric disorders, such as schizophrenia and bipolar disorder. Mice with targeted disruption of the nNOS gene exhibit abnormal behaviors. Here, we subjected nNOS knockout (KO mice to a battery of behavioral tests to further investigate the role of nNOS in neuropsychiatric functions. We also examined the role of nNOS in dopamine/DARPP-32 signaling in striatal slices from nNOS KO mice and the effects of the administration of a dopamine D1 receptor agonist on behavior in nNOS KO mice. Results nNOS KO mice showed hyperlocomotor activity in a novel environment, increased social interaction in their home cage, decreased depression-related behavior, and impaired spatial memory retention. In striatal slices from nNOS KO mice, the effects of a dopamine D1 receptor agonist, SKF81297, on the phosphorylation of DARPP-32 and AMPA receptor subunit GluR1 at protein kinase A sites were enhanced. Consistent with the biochemical results, intraperitoneal injection of a low dose of SKF81297 significantly decreased prepulse inhibition in nNOS KO mice, but not in wild-type mice. Conclusion These findings indicate that nNOS KO upregulates dopamine D1 receptor signaling, and induces abnormal social behavior, hyperactivity and impaired remote spatial memory. nNOS KO mice may serve as a unique animal model of psychiatric disorders.

Multiple sleep alterations in mice lacking cannabinoid type 1 receptors.

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Alessandro Silvani

Full Text Available Cannabinoid type 1 (CB1 receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD. We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD and HFD. CB1 cannabinoid receptor knock-out (KO and wild-type (WT mice were fed SD or HFD for 4 months (n = 9-10 per group. Mice were instrumented with electroencephalographic (EEG and electromyographic electrodes. Recordings were performed during baseline (48 hours, sleep deprivation (gentle handling, 6 hours, sleep recovery (18 hours, and after cage switch (insomnia model paradigm, 6 hours. We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS and rapid-eye-movement sleep (REMS than WT during the dark (active period but not during the light (rest period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice.

ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.).

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Pandeya, Devendra; Campbell, LeAnne M; Nunes, Eugenia; Lopez-Arredondo, Damar L; Janga, Madhusudhana R; Herrera-Estrella, Luis; Rathore, Keerti S

2017-12-01

This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

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

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

2015-01-01

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

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

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

2015-11-01

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. Copyright © 2015. Published by Elsevier Inc.

Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1 (GIT1 knock out mice.

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Guoyong Yin

Full Text Available 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.

Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

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Hwang, M; Go, Y; Park, J-H; Shin, S-K; Song, S E; Oh, B-C; Im, S-S; Hwang, I; Jeon, Y H; Lee, I-K; Seino, S; Song, D-K

2017-02-01

The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic

PET Imaging on Dynamic Metabolic Changes after Combination Therapy of Paclitaxel and the Traditional Chinese Medicine in Breast Cancer-Bearing Mice.

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Chen, Yao; Wang, Ling; Liu, Hao; Song, Fahuan; Xu, Caiyun; Zhang, Kai; Chen, Qing; Wu, Shuang; Zhu, Yunqi; Dong, Ying; Zhou, Min; Zhang, Hong; Tian, Mei

2018-04-01

The aim of the study was to non-invasively evaluate the anticancer activity of a traditional Chinese medicine-Huaier, combined with paclitaxel (PTX) in breast cancer bearing mice by detecting dynamic metabolic changes with positron emission tomography (PET). Balb/c nude mice were randomly divided into one of the four groups: Huaier, PTX, PTX + Huaier, or the control. PET imaging with 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) was performed to monitor the metabolic changes in BT474 (luminal B) and MDA-MB-231 (triple-negative) breast cancer xenografts. Immunohistochemistry (IHC) study was performed immediately after the final PET scan to assess the expressions of phosphatidylinositol 3-kinase (PI3K), phospho-AKT (p-AKT), caspase-3, and vascular endothelial growth factor (VEGF). Compared to the control group, [ 18 F]FDG accumulation demonstrated a significant decrease in PTX + Huaier (p PET imaging could be a potential non-invasive approach to assess the metabolic changes after chemotherapy combined with traditional Chinese medicine in the breast cancer.

Effect of nerve injury on the number of dorsal root ganglion neurons and autotomy behavior in adult Bax-deficient mice

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Lyu C

2017-08-01

Full Text Available Chuang Lyu,1,2 Gong-Wei Lyu,3 Aurora Martinez,4 Tie-Jun Sten Shi4 1State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China; 2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 3Department of Neurology, 1st Hospital of Harbin Medical University, Harbin, People’s Republic of China; 4Department of Biomedicine, University of Bergen, Bergen, Norway Background: The proapoptotic molecule BAX, plays an important role in mitochondrial apoptotic pathway. Dorsal root ganglion (DRG neurons depend on neurotrophic factors for survival at early developmental stages. Withdrawal of neurotrophic factors will induce apoptosis in DRG neurons, but this type of cell death can be delayed or prevented in neonatal Bax knockout (KO mice. In adult animals, evidence also shows that DRG neurons are less dependent upon neurotrophic factors for survival. However, little is known about the effect of Bax deletion on the survival of normal and denervated DRG neurons in adult mice. Methods: A unilateral sciatic nerve transection was performed in adult Bax KO mice and wild-type (WT littermates. Stereological method was employed to quantify the number of lumbar-5 DRG neurons 1 month post-surgery. Nerve injury-induced autotomy behavior was also examined on days 1, 3, and 7 post-surgery. Results: There were significantly more neurons in contralateral DRGs of KO mice as compared with WT mice. The number of neurons was reduced in ipsilateral DRGs in both KO and WT mice. No changes in size distributions of DRG neuron profiles were detected before or after nerve injury. Injury-induced autotomy behavior developed much earlier and was more serious in KO mice. Conclusion: Although postnatal death or loss of DRG neurons is partially prevented by Bax deletion, this effect cannot interfere with long-term nerve injury-induced neuronal loss. The exaggerated self

GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior.

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Aizawa, Fuka; Nishinaka, Takashi; Yamashita, Takuya; Nakamoto, Kazuo; Kurihara, Takashi; Hirasawa, Akira; Kasuya, Fumiyo; Miyata, Atsuro; Tokuyama, Shogo

2016-12-01

The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Distorted leukocyte migration, angiogenesis, wound repair and metastasis in Tspan8 and Tspan8/CD151 double knockout mice indicate complementary activities of Tspan8 and CD51.

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Zhao, Kun; Erb, Ulrike; Hackert, Thilo; Zöller, Margot; Yue, Shijing

2018-02-01

The tetraspanin Tspan8 supports via associated integrins and proteases tumor progression and angiogenesis. To shed light on its activities in non-transformed cells, we generated a Tspan8 knockout (ko) mouse, comparing leukocyte migration, angiogenesis, wound healing and tumor growth with wild type, CD151ko and Tspan8/CD151ko (dbko) mice. CD151ko mice were included as CD151 activities resemble that of Tspan8, and dbko mice to exclude mutual substitution. Tspan8ko and dbko mice show no pathological phenotype. However, delayed type hypersensitivity reactions are mitigated in Tspan8ko mice, angiogenesis is severely impaired in Tspan8ko, CD151ko and dbko mice, with Tspan8 mostly affecting lymphangiogenesis. Distinct contributions of CD151 and Tspan8 to skin wound healing rely on preferentially CD151 anchoring basal keratinocytes and Tspan8 promoting motility. Proliferation of wounded skin keratinocytes is not affected. Metastasis formation of a melanoma and a Tspan8-expressing pancreatic cancer line was impaired in Tspan8ko and dbko mice, pointing towards a contribution of host Tspan8 to tumor progression. In line with the importance of tetraspanins in exosome-mediated intercellular communication, defects became mitigated by Tspan8/CD151-competent serum exosomes, which offers a most promising therapeutic option for chronic wounds and arteriosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Commensal microbiota contributes to chronic endocarditis in TAX1BP1 deficient mice.

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Satoko Nakano

Full Text Available Tax1-binding protein 1 (Tax1bp1 negatively regulates NF-κB by editing the ubiquitylation of target molecules by its catalytic partner A20. Genetically engineered TAX1BP1-deficient (KO mice develop age-dependent inflammatory constitutions in multiple organs manifested as valvulitis or dermatitis and succumb to premature death. Laser capture dissection and gene expression microarray analysis on the mitral valves of TAX1BP1-KO mice (8 and 16 week old revealed 588 gene transcription alterations from the wild type. SAA3 (serum amyloid A3, CHI3L1, HP, IL1B and SPP1/OPN were induced 1,180-, 361-, 187-, 122- and 101-fold respectively. WIF1 (Wnt inhibitory factor 1 exhibited 11-fold reduction. Intense Saa3 staining and significant I-κBα reduction were reconfirmed and massive infiltration of inflammatory lymphocytes and edema formation were seen in the area. Antibiotics-induced 'germ free' status or the additional MyD88 deficiency significantly ameliorated TAX1BP1-KO mice's inflammatory lesions. These pathological conditions, as we named 'pseudo-infective endocarditis' were boosted by the commensal microbiota who are usually harmless by their nature. This experimental outcome raises a novel mechanistic linkage between endothelial inflammation caused by the ubiquitin remodeling immune regulators and fatal cardiac dysfunction.

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

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Harrill, Joshua A. [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States); Hukkanen, Renee R.; Lawson, Marie; Martin, Greg [The Dow Chemical Company, Midland, MI 48640 (United States); Gilger, Brian [North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27606 (United States); Soldatow, Valerie [University of North Carolina, Department of Environmental Sciences and Engineering, Chapel Hill, NC 27599 (United States); LeCluyse, Edward L. [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States); Budinsky, Robert A.; Rowlands, J. Craig [The Dow Chemical Company, Midland, MI 48640 (United States); Thomas, Russell S., E-mail: RThomas@thehamner.org [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States)

2013-10-15

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct

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Pavlov, Tengis S.; Ilatovskaya, Daria V.; Levchenko, Vladislav; Li, Lijun; Ecelbarger, Carolyn M.; Staruschenko, Alexander

2013-01-01

The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.—Pavlov, T. S., Ilatovskaya, D. V., Levchenko, V., Li, L., Ecelbarger, C. M., Staruschenko, A. Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct. PMID:23558339

Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviors in aged mice.

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Dworkin, Sebastian; Auden, Alana; Partridge, Darren D; Daglas, Maria; Medcalf, Robert L; Mantamadiotis, Theo; Georgy, Smitha R; Darido, Charbel; Jane, Stephen M; Ting, Stephen B

2017-06-01

The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation, and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here they show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviors. Using both Grhl3-knockout mice (Grhl3 -/- ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3 flox/flox ), they performed histological expression analyses and behavioral tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety, and stress. They found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain; however, aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviors suggestive of decreased fear and anxiety. They conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behavior or hyperactivity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 775-788, 2017. © 2017 Wiley Periodicals, Inc.

Sortilin 1 Loss-of-Function Protects Against Cholestatic Liver Injury by Attenuating Hepatic Bile Acid Accumulation in Bile Duct Ligated Mice.

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Li, Jibiao; Woolbright, Benjamin L; Zhao, Wen; Wang, Yifeng; Matye, David; Hagenbuch, Bruno; Jaeschke, Hartmut; Li, Tiangang

2018-01-01

Sortilin 1 (Sort1) is an intracellular trafficking receptor that mediates protein sorting in the endocytic or secretory pathways. Recent studies revealed a role of Sort1 in the regulation of cholesterol and bile acid (BA) metabolism. This study further investigated the role of Sort1 in modulating BA detoxification and cholestatic liver injury in bile duct ligated mice. We found that Sort1 knockout (KO) mice had attenuated liver injury 24 h after bile duct ligation (BDL), which was mainly attributed to less bile infarct formation. Sham-operated Sort1 KO mice had about 20% larger BA pool size than sham-operated wildtype (WT) mice, but 24 h after BDL Sort1 KO mice had significantly attenuated hepatic BA accumulation and smaller BA pool size. After 14 days BDL, Sort1 KO mice showed significantly lower hepatic BA concentration and reduced expression of inflammatory and fibrotic marker genes, but similar degree of liver fibrosis compared with WT mice. Unbiased quantitative proteomics revealed that Sort1 KO mice had increased hepatic BA sulfotransferase 2A1, but unaltered phase-I BA metabolizing cytochrome P450s or phase-III BA efflux transporters. Consistently, Sort1 KO mice showed elevated plasma sulfated taurocholate after BDL. Finally, we found that liver Sort1 was repressed after BDL, which may be due to BA activation of farnesoid x receptor. In conclusion, we report a role of Sort1 in the regulation of hepatic BA detoxification and cholestatic liver injury in mice. The mechanisms underlying increased hepatic BA elimination in Sort1 KO mice after BDL require further investigation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Loss of epithelial FAM20A in mice causes amelogenesis imperfecta, tooth eruption delay and gingival overgrowth.

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Li, Li-Li; Liu, Pei-Hong; Xie, Xiao-Hua; Ma, Su; Liu, Chao; Chen, Li; Qin, Chun-Lin

2016-06-30

FAM20A has been studied to a very limited extent. Mutations in human FAM20A cause amelogenesis imperfecta, gingival fibromatosis and kidney problems. It would be desirable to systemically analyse the expression of FAM20A in dental tissues and to assess the pathological changes when this molecule is specifically nullified in individual tissues. Recently, we generated mice with a Fam20A-floxed allele containing the beta-galactosidase reporter gene. We analysed FAM20A expression in dental tissues using X-Gal staining, immunohistochemistry and in situ hybridization, which showed that the ameloblasts in the mouse mandibular first molar began to express FAM20A at 1 day after birth, and the reduced enamel epithelium in erupting molars expressed a significant level of FAM20A. By breeding K14-Cre mice with Fam20A(flox/flox) mice, we created K14-Cre;Fam20A(flox/flox) (conditional knock out, cKO) mice, in which Fam20A was inactivated in the epithelium. We analysed the dental tissues of cKO mice using X-ray radiography, histology and immunohistochemistry. The molar enamel matrix in cKO mice was much thinner than normal and was often separated from the dentinoenamel junction. The Fam20A-deficient ameloblasts were non-polarized and disorganized and were detached from the enamel matrix. The enamel abnormality in cKO mice was consistent with the diagnosis of amelogenesis imperfecta. The levels of enamelin and matrix metalloproteinase 20 were lower in the ameloblasts and enamel of cKO mice than the normal mice. The cKO mice had remarkable delays in the eruption of molars and hyperplasia of the gingival epithelium. The findings emphasize the essential roles of FAM20A in the development of dental and oral tissues.

Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

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Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni

2005-10-01

We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

Deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 knockout mice.

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Zhang, Danqing; Kobayashi, Toshiyuki; Kojima, Tetsuo; Kanenishi, Kenji; Hagiwara, Yoshiaki; Abe, Masaaki; Okura, Hidehiro; Hamano, Yoshitomo; Sun, Guodong; Maeda, Masahiro; Jishage, Kou-ichi; Noda, Tetsuo; Hino, Okio

2011-04-01

Genetic crossing experiments were performed between tuberous sclerosis-2 (Tsc2) KO and expressed in renal carcinoma (Erc) KO mice to analyze the function of the Erc/mesothelin gene in renal carcinogenesis. We found the number and size of renal tumors were significantly less in Tsc2+/-;Erc-/- mice than in Tsc2+/-;Erc+/+ and Tsc2+/-;Erc+/- mice. Tumors from Tsc2+/-;Erc-/- mice exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen (Ki67) and TUNEL analysis, respectively. Adhesion to collagen-coated plates in vitro was enhanced in Erc-restored cells and decreased in Erc-suppressed cells with siRNA. Tumor formation by Tsc2-deficient cells in nude mice was remarkably suppressed by stable knockdown of Erc with shRNA. Western blot analysis showed that the phosphorylation of focal adhesion kinase, Akt and signal transducer and activator of transcription protein 3 were weaker in Erc-deficient/suppressed cells compared with Erc-expressed cells. These results indicate that deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 KO mice and inhibits the phosphorylation of several kinases of cell adhesion mechanism. This suggests that Erc/mesothelin may have an important role in the promotion and/or maintenance of carcinogenesis by influencing cell-substrate adhesion via the integrin-related signal pathway. © 2011 Japanese Cancer Association.

NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

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Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C; Dominik, Malte J; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas; Steinmetz, Ivo

2017-02-01

Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91 phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91 phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91 phox KO mice. Only infected gp91 phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis. Copyright © 2017 American Society for Microbiology.

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

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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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effects of Nrf2 deficiency on arsenic metabolism in mice.

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Wang, Huihui; Zhu, Jiayu; Li, Lu; Li, Yongfang; Lv, Hang; Xu, Yuanyuan; Sun, Guifan; Pi, Jingbo

2017-12-15

Inorganic arsenic (iAs) is a known toxicant and carcinogen. Worldwide arsenic exposure has become a threat to human health. The severity of arsenic toxicity is strongly correlated with the speed of arsenic metabolism (methylation) and clearance. Furthermore, oxidative stress is recognized as a major mechanism for arsenic-induced toxicity. Nuclear factor-E2-related factor 2 (Nrf2), a key regulator in cellular adaptive antioxidant response, is clearly involved in alleviation of arsenic-induced oxidative damage. Multiple studies demonstrate that Nrf2 deficiency mice are more vulnerable to arsenic-induced intoxication. However, what effect Nrf2 deficiency might have on arsenic metabolism in mice is still unknown. In the present study, we measured the key enzymes involved in arsenic metabolism in Nrf2-WT and Nrf2-KO mice. Our results showed that basal transcript levels of glutathione S-transferase omega 2 (Gsto2) were significantly higher and GST mu 1 (Gstm1) lower in Nrf2-KO mice compared to Nrf2-WT control. Arsenic speciation and methylation rate in liver and urine was then studied in mice treated with 5mg/kg sodium arsenite for 12h. Although there were some alterations in arsenic metabolism enzymes between Nrf2-WT and Nrf2-KO mice, the Nrf2 deficiency had no significant effect on arsenic methylation. These results suggest that the Nrf2-KO mice are more sensitive to arsenic than Nrf2-WT mainly because of differences in adaptive antioxidant detoxification capacity rather than arsenic methylation capacity. Copyright © 2017. Published by Elsevier Inc.

Hippocampal network oscillations in APP/APLP2-deficient mice.

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Xiaomin Zhang

Full Text Available The physiological function of amyloid precursor protein (APP and its two homologues APP-like protein 1 (APLP1 and 2 (APLP2 is largely unknown. Previous work suggests that lack of APP or APLP2 impairs synaptic plasticity and spatial learning. There is, however, almost no data on the role of APP or APLP at the network level which forms a critical interface between cellular functions and behavior. We have therefore investigated memory-related synaptic and network functions in hippocampal slices from three lines of transgenic mice: APPsα-KI (mice expressing extracellular fragment of APP, corresponding to the secreted APPsα ectodomain, APLP2-KO, and combined APPsα-KI/APLP2-KO (APPsα-DM for "double mutants". We analyzed two prominent patterns of network activity, gamma oscillations and sharp-wave ripple complexes (SPW-R. Both patterns were generally preserved in all strains. We find, however, a significantly reduced frequency of gamma oscillations in CA3 of APLP2-KO mice in comparison to APPsα-KI and WT mice. Network activity, basic synaptic transmission and short-term plasticity were unaltered in the combined mutants (APPsα-DM which showed, however, reduced long-term potentiation (LTP. Together, our data indicate that APLP2 and the intracellular domain of APP are not essential for coherent activity patterns in the hippocampus, but have subtle effects on synaptic plasticity and fine-tuning of network oscillations.

Salt-Sensitive Hypertension and Cardiac Hypertrophy in Transgenic Mice Expressing a Corin Variant Identified in African Americans

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Wang, Wei; Cui, Yujie; Shen, Jianzhong; Jiang, Jingjing; Chen, Shenghan; Peng, Jianhao; Wu, Qingyu

2012-01-01

African Americans represent a high risk population for salt-sensitive hypertension and heart disease but the underlying mechanism remains unclear. Corin is a cardiac protease that regulates blood pressure by activating natriuretic peptides. A corin gene variant (T555I/Q568P) was identified in African Americans with hypertension and cardiac hypertrophy. In this study, we test the hypothesis that the corin variant contributes to the hypertensive and cardiac hypertrophic phenotype in vivo. Transgenic mice were generated to express wild-type or T555I/Q568P variant corin in the heart under the control of α-myosin heavy chain promoter. The mice were crossed into a corin knockout background to create KO/TgWT and KO/TgV mice that expressed WT or variant corin, respectively, in the heart. Functional studies showed that KO/TgV mice had significantly higher levels of pro-atrial natriuretic peptide in the heart compared with that in control KO/TgWT mice, indicating that the corin variant was defective in processing natriuretic peptides in vivo. By radiotelemetry, corin KO/TgV mice were found to have hypertension that was sensitive to dietary salt loading. The mice also developed cardiac hypertrophy at 12–14 months of age when fed a normal salt diet or at a younger age when fed a high salt diet. The phenotype of salt-sensitive hypertension and cardiac hypertrophy in KO/TgV mice closely resembles the pathological findings in African Americans who carry the corin variant. The results indicate that corin defects may represent an important mechanism in salt-sensitive hypertension and cardiac hypertrophy in African Americans. PMID:22987923

A strong anti-inflammatory signature revealed by liver transcription profiling of Tmprss6-/- mice.

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Michela Riba

Full Text Available Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe(-/- mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe(-/- deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6.

TLR3 signaling is either protective or pathogenic for the development of Theiler's virus-induced demyelinating disease depending on the time of viral infection

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Jin Young-Hee

2011-12-01

Full Text Available Abstract Background We have previously shown that toll-like receptor 3 (TLR3-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler's murine encephalomyelitis virus (TMEV infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease. Methods SJL/J and B6;129S-Tlr3tm1Flv/J (TLR3KO-B6 mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theiler's murine encephalomyelitis virus (2 × 105 PFU with or without treatment with 50 μg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed. Results We investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and

Treatment with the GSK3-beta inhibitor Tideglusib improves hippocampal development and memory performance in juvenile, but not adult, Cdkl5 knockout mice.

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Fuchs, Claudia; Fustini, Norma; Trazzi, Stefania; Gennaccaro, Laura; Rimondini, Roberto; Ciani, Elisabetta

2018-05-01

Cyclin-dependent kinase-like 5 (CDKL5) disorder is a severe neurodevelopmental disorder characterized by early-onset epileptic seizures, severe developmental delay, and intellectual disability. To date, no effective pharmacological treatments are available to improve the neurological phenotype that is due to mutations in the CDKL5 gene. Murine models of CDKL5 disorder have recently been generated, making the preclinical testing of pharmacological interventions possible. Using a Cdkl5 knockout (KO) mouse model, we recently demonstrated that deficiency of Cdkl5 causes defects in postnatal hippocampal development and hippocampus-dependent learning and memory. These defects were accompanied by an increased activity of GSK3β, an important inhibitory regulator of many neuronal functions. Pharmacological inhibition of GSK3β activity was able to recover hippocampal defects and cognitive performance in juvenile Cdkl5 KO mice, suggesting that GSK3β inhibitors might be a potential therapeutic option for CDKL5 disorder. As GSK3β inhibitors have been shown to have differential medication responses in young people and adults, this study was designed to examine whether GSK3β is a possible therapeutic target both in juvenile and in adult CDKL5 patients. We found that treatment with the GSK3β inhibitor Tideglusib during the juvenile period improved hippocampal development and hippocampus-dependent behaviors in Cdkl5 KO mice, while treatment later on in adulthood had no positive effects. These results suggest that pharmacological interventions aimed at normalizing impaired GSK3β activity might have different age-dependent outcomes in CDKL5 disorder. This is of utmost importance in the development of therapeutic approaches in CDKL5 patients and in the design of rational clinical trials. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets.

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Du Toit, Eugene; Browne, Liam; Irving-Rodgers, Helen; Massa, Helen M; Fozzard, Nicolette; Jennings, Michael P; Peak, Ian R

2017-04-20

Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the aetiology of obesity and glucose intolerance is not well characterised. We set out to determine whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body weight when comparing WT and GPR84 KO mice on the respective diets. Glucose tolerance was also similar in WT and GPR84 KO mice irrespective of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. GPR84 deletion had no effects on body weight or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metabolism, as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

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Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

Isolation of a third species of Sarcocystis in immunodeficient mice fed feces from opossums (Didelphis virginiana) and its differentiation from Sarcocystis falcatula and Sarcocystis neurona.

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Dubey, J P; Speer, C A; Lindsay, D S

1998-12-01

Opossums (Didelphis virginiana) were found to be hosts for 3 species of Sarcocystis: Sarcocystis falcatula with an avian intermediate host, S. neurona with an undetermined intermediate host, and a third, unnamed, species. Sporocysts from the intestines of 2 opossums (nos. 26 and 47) were fed to budgerigars (Melopsittacus undulatus), nude mice, and gamma-interferon knockout (KO) mice. Sporocysts of S. falcatula were not infective to nude or KO mice. Sporocysts of S. neurona induced encephalitis in KO and nude mice; only schizonts and merozoites were found in tissues of mice, and they reacted with anti-S. neurona serum raised against the SN-2 isolate of S. neurona originally obtained from tissues of a paralyzed horse. All 3 species of Sarcocystis were present in opossum no. 47. Sarcocystis neurona was isolated in cell culture from this opossum. Sporocysts from opossum no. 47 were lethal to budgerigars, indicating S. falcatula infection. Only 1 species of Sarcocystis (the third species) was found in opossum no. 26; the sporocysts were infective to KO and nude mice. Schizonts and merozoites of this species were predominantly in the liver but were also found in other tissues; schizonts did not react with anti-S. neurona serum. Merozoites of the third species were ultrastructurally distinct from S. falcatula and S. neurona merozoites. Sarcocysts were found in leg muscles of 2 mice killed 50 and 54 days after they were fed sporocysts from opossum no. 26. These sarcocysts had steeple-shaped protrusions on the cyst wall and were distinct from sarcocysts of S. falcatula and any other species of Sarcocystis.

CD44 and Bak expression in IL-6 or TNF-alpha gene knockout mice after whole lung irradiation

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Sakai, Minako; Iwakawa, Mayumi; Ohta, Toshie; Tsujii, Hirohiko; Imai, Takashi; Iwakura, Yoichiro

2008-01-01

To understand the molecular mechanisms that underlie radiation pneumonitis, we examined whether knockout of the tumor necrosis factor (TNF) or the interleukin (IL)-6 gene could give mice an inherent resistance to radiation in the acute phase of alveolar damage after thoracic irradiation. The temporal expression of inflammation (CD44) and apoptosis (Bak) markers in lung after thoracic irradiation was measured to determine the degree of alveolar damage. At 4 weeks post-irradiation (10 Gy), small inflammatory foci were observed in all mice, but there were no obvious histological differences between control (C57BL/6JSlc), TNF-alpha knockout (TNF KO), and IL-6 knockout (IL-6 KO) mice. However, immunohistochemical analysis of CD44 and Bak expression over a time course of 2 weeks highlighted significant differences between the three groups. C57BL/6JSlc and TNF KO mice had increased numbers of both CD44-positive and Bak-positive cells after irradiation, while the IL-6 KO mice showed stable levels of CD44 and Bak. In conclusion, the radioresistant status of IL-6 KO mice in the acute phase of alveolar damage after irradiation suggested an important role for IL-6 in radiation pneumonitis. (author)

A pathophysiological role of TRPV1 in ischemic injury after transient focal cerebral ischemia in mice

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Miyanohara, Jun [Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University (Japan); Shirakawa, Hisashi, E-mail: shirakaw@pharm.kyoto-u.ac.jp [Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University (Japan); Sanpei, Kazuaki [Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University (Japan); Nakagawa, Takayuki [Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University (Japan); Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital (Japan); Kaneko, Shuji [Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University (Japan)

2015-11-20

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with high Ca{sup 2+} permeability, which functions as a polymodal nociceptor activated by heat, protons and several vanilloids, including capsaicin and anandamide. Although TRPV1 channels are widely distributed in the mammalian brain, their pathophysiological roles in the brain remain to be elucidated. In this study, we investigated whether TRPV1 is involved in cerebral ischemic injury using a middle cerebral artery (MCA) occlusion model in wild-type (WT) and TRPV1-knockout (KO) mice. For transient ischemia, the left MCA of C57BL/6 mice was occluded for 60 min and reperfused at 1 and 2 days after ischemia. We found that neurological and motor deficits, and infarct volumes in TRPV1-KO mice were lower than those of WT mice. Consistent with these results, intracerebroventricular injection of a TRPV1 antagonist, capsazepine (20 nmol), 30 min before the onset of ischemia attenuated neurological and motor deficits and improved infarct size without influencing cerebral blood flow in the occluded MCA territory. The protective effect of capsazepine on ischemic brain damage was not observed in TRPV1-KO mice. WT and TRPV1-KO mice did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 2 days after cerebral ischemia. Taken together, we conclude that brain TRPV1 channels are activated by ischemic stroke and cause neurological and motor deficits and infarction after brain ischemia. - Highlights: • We investigated whether TRPV1 is involved in transient ischemic brain damage in mice. • Neurological deficits and infarct volumes were lower in TRPV1-KO mice than in WT mice. • Injection of a TRPV1 antagonist, capsazepine, attenuated neurological deficits and improved infarct size. • No differences in astrocytic or microglial activation were observed between WT and TRPV1-KO mice.

A pathophysiological role of TRPV1 in ischemic injury after transient focal cerebral ischemia in mice

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Miyanohara, Jun; Shirakawa, Hisashi; Sanpei, Kazuaki; Nakagawa, Takayuki; Kaneko, Shuji

2015-01-01

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with high Ca"2"+ permeability, which functions as a polymodal nociceptor activated by heat, protons and several vanilloids, including capsaicin and anandamide. Although TRPV1 channels are widely distributed in the mammalian brain, their pathophysiological roles in the brain remain to be elucidated. In this study, we investigated whether TRPV1 is involved in cerebral ischemic injury using a middle cerebral artery (MCA) occlusion model in wild-type (WT) and TRPV1-knockout (KO) mice. For transient ischemia, the left MCA of C57BL/6 mice was occluded for 60 min and reperfused at 1 and 2 days after ischemia. We found that neurological and motor deficits, and infarct volumes in TRPV1-KO mice were lower than those of WT mice. Consistent with these results, intracerebroventricular injection of a TRPV1 antagonist, capsazepine (20 nmol), 30 min before the onset of ischemia attenuated neurological and motor deficits and improved infarct size without influencing cerebral blood flow in the occluded MCA territory. The protective effect of capsazepine on ischemic brain damage was not observed in TRPV1-KO mice. WT and TRPV1-KO mice did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 2 days after cerebral ischemia. Taken together, we conclude that brain TRPV1 channels are activated by ischemic stroke and cause neurological and motor deficits and infarction after brain ischemia. - Highlights: • We investigated whether TRPV1 is involved in transient ischemic brain damage in mice. • Neurological deficits and infarct volumes were lower in TRPV1-KO mice than in WT mice. • Injection of a TRPV1 antagonist, capsazepine, attenuated neurological deficits and improved infarct size. • No differences in astrocytic or microglial activation were observed between WT and TRPV1-KO mice.

Treatment with the anti-IL-6 receptor antibody attenuates muscular dystrophy via promoting skeletal muscle regeneration in dystrophin-/utrophin-deficient mice.

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Wada, Eiji; Tanihata, Jun; Iwamura, Akira; Takeda, Shin'ichi; Hayashi, Yukiko K; Matsuda, Ryoichi

2017-10-27

Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice. Male dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age. Treatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody. As no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy

Elastase-2, a Tissue Alternative Pathway for Angiotensin II Generation, Plays a Role in Circulatory Sympathovagal Balance in Mice.

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Becari, Christiane; Durand, Marina T; Guimaraes, Alessander O; Lataro, Renata M; Prado, Cibele M; de Oliveira, Mauro; Candido, Sarai C O; Pais, Paloma; Ribeiro, Mauricio S; Bader, Michael; Pesquero, Joao B; Salgado, Maria C O; Salgado, Helio C

2017-01-01

In vitro and ex vivo experiments indicate that elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, is an alternative pathway for angiotensin II (Ang II) generation. However, the role played by ELA-2 in vivo is unclear. We examined ELA-2 knockout (ELA-2KO) mice compared to wild-type (WT) mice and determined whether ELA-2 played a role in hemodynamics [arterial pressure (AP) and heart rate (HR)], cardiocirculatory sympathovagal balance and baroreflex sensitivity. The variability of systolic arterial pressure (SAP) and pulse interval (PI) for evaluating autonomic modulation was examined for time and frequency domains (spectral analysis), whereas a symbolic analysis was also used to evaluate PI variability. In addition, baroreflex sensitivity was examined using the sequence method. Cardiac function was evaluated echocardiographically under anesthesia. The AP was normal whereas the HR was reduced in ELA-2KO mice (425 ± 17 vs. 512 ± 13 bpm from WT). SAP variability and baroreflex sensitivity were similar in both strains. The LF power from the PI spectrum (33.6 ± 5 vs. 51.8 ± 4.8 nu from WT) and the LF/HF ratio (0.60 ± 0.1 vs. 1.45 ± 0.3 from WT) were reduced, whereas the HF power was increased (66.4 ± 5 vs. 48.2 ± 4.8 nu from WT) in ELA-2KO mice, indicating a shift toward parasympathetic modulation of HR. Echocardiographic examination showed normal fractional shortening and an ejection fraction in ELA-2KO mice; however, the cardiac output, stroke volume, and ventricular size were reduced. These findings provide the first evidence that ELA-2 acts on the sympathovagal balance of the heart, as expressed by the reduced sympathetic modulation of HR in ELA-2KO mice.

Lycopene attenuated hepatic tumorigenesis via differential mechanisms depending on carotenoid cleavage enzyme in mice

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Ip, Blanche C.; Liu, Chun; Ausman, Lynne M.; von Lintig, Johannes; Wang, Xiang-Dong

2014-01-01

Obesity is associated with increased liver cancer risks and mortality. We recently showed that apo-10’-lycopenoic acid, a lycopene metabolite generated by beta-carotene-9’,10’-oxygenase (BCO2), inhibited carcinogen-initiated, high-fat diet (HFD)-promoted liver inflammation and hepatic tumorigenesis development. The present investigation examined the outstanding question of whether the lycopene could suppress HFD-promoted hepatocellular carcinoma (HCC) progression, and if BCO2 is important in BCO2-knockout (BCO2-KO) and wild-type male mice. Results showed that lycopene supplementation (100 mg/kg diet) for 24 weeks resulted in comparable accumulation of hepatic lycopene (19.4 vs 18.2 nmol/g) and had similar effects on suppressing HFD-promoted HCC incidence (19% vs 20%) and multiplicity (58% vs 62%) in wild-type and BCO2-KO mice, respectively. Intriguingly, lycopene chemopreventive effects in wild-type mice were associated with reduced hepatic pro-inflammatory signaling (phosphorylation of nuclear factor-κB p65 and signal transducer and activator of transcription 3; interleukin-6 protein) and inflammatory foci. In contrast, the protective effects of lycopene in BCO2-KO but not in wild-type mice were associated with reduced hepatic endoplasmic reticulum stress-mediated unfolded protein response (ERUPR), through decreasing ERUPR-mediated protein kinase RNA-activated like kinase– eukaryotic initiation factor 2α activation, and inositol requiring 1α–X-box binding protein 1 signaling. Lycopene supplementation in BCO2-KO mice suppressed oncogenic signals including Met mRNA, β-catenin protein, and mammalian target of rapamycin (mTOR) complex 1 activation, which was associated with increased hepatic microRNA (miR)-199a/b and miR-214 levels. These results provided novel experimental evidence that dietary lycopene can prevent HFD-promoted HCC incidence and multiplicity in mice, and may elicit different mechanisms depending on BCO2 expression. PMID:25293877

Rasgrf2 controls dopaminergic adaptations to alcohol in mice.

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Easton, Alanna C; Rotter, Andrea; Lourdusamy, Anbarasu; Desrivières, Sylvane; Fernández-Medarde, Alberto; Biermann, Teresa; Fernandes, Cathy; Santos, Eugenio; Kornhuber, Johannes; Schumann, Gunter; Müller, Christian P

2014-10-01

Alcohol abuse leads to serious health problems with no effective treatment available. Recent evidence suggests a role for ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) in alcoholism. Rasgrf2 is a calcium sensor and MAPK/ERK activating protein, which has been linked to neurotransmitter release and monoaminergic receptor adaptations. Rasgrf2 knock out (KO) mice do not develop a dopamine response in the nucleus accumbens after an alcohol challenge and show a reduced consumption of alcohol. The present study aims to further characterise the role of Rasgrf2 in dopaminergic activation beyond the nucleus accumbens following alcohol treatment. Using in vivo microdialysis we found that alcohol induces alterations in dopamine levels in the dorsal striatum between wildtype (WT) and Rasgrf2 KO mice. There was no difference in the expression of dopamine transporter (DAT), dopamine receptor regulating factor (DRRF), or dopamine D2 receptor (DRD2) mRNA in the brain between Rasgrf2 KO and WT mice. After sub-chronic alcohol treatment, DAT and DRRF, but not DRD2 mRNA expression differed between WT and Rasgrf2 KO mice. Brain adaptations were positively correlated with splenic expression levels. These data suggest that Rasgrf2 controls dopaminergic signalling and adaptations to alcohol also in other brain regions, beyond the nucleus accumbens. Copyright © 2014 Elsevier Inc. All rights reserved.

Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors.

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Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K

2008-09-01

Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive

Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway.

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Hadden, Coedy; Fahmi, Tariq; Cooper, Anthonya; Savenka, Alena V; Lupashin, Vladimir V; Roberts, Drucilla J; Maroteaux, Luc; Hauguel-de Mouzon, Sylvie; Kilic, Fusun

2017-12-01

Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO, and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway. © 2017 Wiley Periodicals, Inc.

p21WAF1/Cip1/Sdi1 knockout mice respond to doxorubicin with reduced cardiotoxicity

International Nuclear Information System (INIS)

Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho; Williams, Stuart; Chen, Qin M.

2011-01-01

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21 WAF1/Cip1/Sdi1 (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: ► Doxorubicin induces p21 elevation in the myocardium. ► Doxorubicin causes dilated cardiomyopathy in wild type mice. ► p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. ► Lack of inflammatory response correlates with the resistance in p21 knockout mice.

Novel approach for a PTX/VEGF dual drug delivery system in cardiovascular applications-an innovative bulk and surface drug immobilization.

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Wulf, Katharina; Teske, Michael; Matschegewski, Claudia; Arbeiter, Daniela; Bajer, Dalibor; Eickner, Thomas; Schmitz, Klaus-Peter; Grabow, Niels

2018-06-01

The successive incorporation of several drugs into the polymeric bulk of implants mostly results in loss of considerable quantity of one drug, and/or the loss in quality of the coating and also in changes of drug release time points. A dual drug delivery system (DDDS) based on poly-L-lactide (PLLA) copolymers combining the effective inhibition of smooth muscle cell proliferation while simultaneously promoting re-endothelialization was successfully developed. To overcome possible antagonistic drug interactions and the limitation of the polymeric bulk material as release system for dual drugs, a novel concept which combines the bulk and surface drug immobilization for a DDDS was investigated. The advantage of this DDDS is that the bulk incorporation of fluorescein diacetate (FDAc) (model drug for paclitaxel (PTX)) via spray coating enhanced the subsequent cleavable surface coupling of vascular endothelial growth factor (VEGF) via the crosslinker bissulfosuccinimidyl suberate (BS 3 ). In the presence of the embedded FDAc, the VEGF loading and release are about twice times higher than in absence. Furthermore, the DDDS combines the diffusion drug delivery (FDAc or PTX) and the chemical controlled drug release, VEGF via hydrolysable ester bonds, without loss in quantity and quality of the drug release curves. Additionally, the performed in vitro biocompatibility study showed the bimodal influences of PTX and VEGF on human endothelial EA.hy926 cells. In conclusion, it was possible to show the feasibility to develop a novel DDDS which has a high potential for the medical application due to the possible easy and short modification of a polymer-based PTX delivery system.

Impaired associative fear learning in mice with complete loss or haploinsufficiency of AMPA GluR1 receptors

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Michael Feyder

2007-12-01

Full Text Available There is compelling evidence that L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA glutamate receptors containing the GluR1 subunit contribute to the molecular mechanisms associated with learning. AMPA GluR1 glutamate receptor knockout mice (KO exhibit abnormal hippocampal and amygdala plasticity, and deficits on various assays for cognition including Pavlovian fear conditioning. Here we examined associative fear learning in mice with complete absence (KO or partial loss (heterozygous mutant, HET of GluR1 on multiple fear conditioning paradigms. After multi-trial delay or trace conditioning, KO displayed impaired tone and context fear recall relative to WT, whereas HET were normal. After one-trial delay conditioning, both KO and HET showed impaired tone and context recall. HET and KO showed normal nociceptive sensitivity in the hot plate and tail flick tests. These data demonstrate that the complete absence of GluR1 subunit-containing receptors prevents the formation of associative fear memories, while GluR1 haploinsufficiency is sufficient to impair one-trial fear learning. These findings support growing evidence of a major role for GluR1-containing AMPA receptors in amygdalamediated forms of learning and memory.

CDKL5 deficiency entails sleep apneas in mice.

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Lo Martire, Viviana; Alvente, Sara; Bastianini, Stefano; Berteotti, Chiara; Silvani, Alessandro; Valli, Alice; Viggiano, Rocchina; Ciani, Elisabetta; Zoccoli, Giovanna

2017-08-01

A recently discovered neurodevelopmental disorder caused by the mutation of the cyclin-dependent kinase-like 5 gene (CDKL5) entails complex autistic-like behaviours similar to Rett syndrome, but its impact upon physiological functions remains largely unexplored. Sleep-disordered breathing is common and potentially life-threatening in patients with Rett syndrome; however, evidence is limited in children with CDKL5 disorder, and is lacking altogether in adults. The aim of this study was to test whether the breathing pattern during sleep differs between adult Cdkl5 knockout (Cdkl5-KO) and wild-type (WT) mice. Using whole-body plethysmography, sleep and breathing were recorded non-invasively for 8 h during the light period. Sleep apneas occurred more frequently in Cdkl5-KO than in WT mice. A receiver operating characteristic (ROC) analysis discriminated Cdkl5-KO significantly from WT mice based on sleep apnea occurrence. These data demonstrate that sleep apneas are a core feature of CDKL5 disorder and a respiratory biomarker of CDKL5 deficiency in mice, and suggest that sleep-disordered breathing should be evaluated routinely in CDKL5 patients. © 2017 European Sleep Research Society.

Phosphodiesterase-1b (Pde1b) knockout mice are resistant to forced swim and tail suspension induced immobility and show upregulation of Pde10a.

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Hufgard, Jillian R; Williams, Michael T; Skelton, Matthew R; Grubisha, Olivera; Ferreira, Filipa M; Sanger, Helen; Wright, Mary E; Reed-Kessler, Tracy M; Rasmussen, Kurt; Duman, Ronald S; Vorhees, Charles V

2017-06-01

Major depressive disorder is a leading cause of suicide and disability. Despite this, current antidepressants provide insufficient efficacy in more than 60% of patients. Most current antidepressants are presynaptic reuptake inhibitors; postsynaptic signal regulation has not received as much attention as potential treatment targets. We examined the effects of disruption of the postsynaptic cyclic nucleotide hydrolyzing enzyme, phosphodiesterase (PDE) 1b, on depressive-like behavior and the effects on PDE1B protein in wild-type (WT) mice following stress. Littermate knockout (KO) and WT mice were tested in locomotor activity, tail suspension (TST), and forced swim tests (FST). FST was also used to compare the effects of two antidepressants, fluoxetine and bupropion, in KO versus WT mice. Messenger RNA (mRNA) expression changes were also determined. WT mice underwent acute or chronic stress and markers of stress and PDE1B expression were examined. Pde1b KO mice exhibited decreased TST and FST immobility. When treated with antidepressants, both WT and KO mice showed decreased FST immobility and the effect was additive in KO mice. Mice lacking Pde1b had increased striatal Pde10a mRNA expression. In WT mice, acute and chronic stress upregulated PDE1B expression while PDE10A expression was downregulated after chronic but not acute stress. PDE1B is a potential therapeutic target for depression treatment because of the antidepressant-like phenotype seen in Pde1b KO mice.

Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice.

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Zheng, Lingjie; Sharma, Rahul; Kung, John T; Deshmukh, Umesh S; Jarjour, Wael N; Fu, Shu Man; Ju, Shyr-Te

2008-04-01

We hypothesize that regulatory T-cell (Treg)-deficient strains have an altered TCR repertoire in part due to the expansion of autoimmune repertoire by self-antigen. We compared the Vbeta family expression profile between B6 and Treg-lacking B6.Cg-Foxp3(sf)(/Y) (B6.sf) mice using fluorescent anti-Vbeta mAbs and observed no changes. However, while the spectratypes of 20 Vbeta families among B6 mice were highly similar, the Vbeta family spectratypes of B6.sf mice were remarkably different from B6 mice and from each other. Significant spectratype changes in many Vbeta families were also observed in Treg-deficient IL-2 knockout (KO) and IL-2Ralpha KO mice. Such changes were not observed with anti-CD3 mAb-treated B6 mice or B6 CD4+CD25- T cells. TCR transgenic (OT-II.sf) mice displayed dramatic reduction of clonotypic TCR with concomitant increase in T cells bearing non-transgenic Vbeta and Valpha families, including T cells with dual receptors expressing reduced levels of transgenic Valpha and endogenous Valpha. Collectively, the data demonstrate that Treg deficiency allows polyclonal expansion of T cells in a stochastic manner, resulting in widespread changes in the TCR repertoire.

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism.

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Xu, Minjun; Kitaura, Yasuyuki; Ishikawa, Takuya; Kadota, Yoshihiro; Terai, Chihaya; Shindo, Daichi; Morioka, Takashi; Ota, Miki; Morishita, Yukako; Ishihara, Kengo; Shimomura, Yoshiharu

2017-01-01

It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism.

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Minjun Xu

Full Text Available It is known that the catabolism of branched-chain amino acids (BCAAs in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK. In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.

Hypothalamic-pituitary thyroid axis alterations in female mice with deletion of the neuromedin B receptor gene.

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Oliveira, Karen J; Paula, Gabriela S M; Império, Guinever E; Bressane, Nina O; Magalhães, Carolina M A; Miranda-Alves, Leandro; Ortiga-Carvalho, Tania M; Pazos-Moura, Carmen C

2014-11-01

Neuromedin B, a peptide highly expressed at the pituitary, has been shown to act as autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here we studied the thyroid axis of adult female mice lacking neuromedin B receptor (NBR-KO), compared to wild type (WT) littermates. They exhibited slight increase in serum TSH (18%), with normal pituitary expression of mRNA coding for α-glycoprotein subunit (Cga), but reduced TSH β-subunit mRNA (Tshb, 41%), lower intra-pituitary TSH content (24%) and increased thyroid hormone transporter MCT-8 (Slc16a2, 44%) and thyroid hormone receptor β mRNA expression (Thrb, 39%). NBR-KO mice exhibited normal thyroxine (T4) and reduced triiodothyronine (T3) (30%), with no alterations in the intra-thyroidal content of T4 and T3 or thyroid morphological changes. Hypothalamic thyrotropin-releasing hormone (TRH) mRNA (Trh) was increased (68%), concomitant with a reduction in type 2 deiodinase mRNA (Dio2, 30%) and no changes in MCT-8 and thyroid hormone receptor mRNA expression. NBR-KO mice exhibited a 56% higher increase in serum TSH in response to an acute single intraperitoneal injection of TRH concomitant with a non-significant increase in pituitary TRH receptor (Trhr) mRNA at basal state. The phenotype of female NBR-KO mice at the hypothalamus-pituitary axis revealed alterations in pituitary and hypothalamic gene expression, associated with reduced serum T3, and higher TSH response to TRH, with apparently normal thyroid morphology and hormonal production. Thus, results confirm that neuromedin B pathways are importantly involved in secretory pathways of TSH and revealed its participation in the in vivo regulation of gene expression of TSH β-subunit and pituitary MCT8 and Thrb and hypothalamic TRH and type 2 deiodinase. Copyright © 2014 Elsevier B.V. All rights reserved.

Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

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

2009-01-01

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

Characterization of intracellular inclusions in the urothelium of mice exposed to inorganic arsenic.

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Dodmane, Puttappa R; Arnold, Lora L; Muirhead, David E; Suzuki, Shugo; Yokohira, Masanao; Pennington, Karen L; Dave, Bhavana J; Lu, Xiufen; Le, X Chris; Cohen, Samuel M

2014-01-01

Inorganic arsenic (iAs) is a known human carcinogen at high exposures, increasing the incidences of urinary bladder, skin, and lung cancers. In most mammalian species, ingested iAs is excreted mainly through urine primarily as dimethylarsinic acid (DMA(V)). In wild-type (WT) mice, iAs, DMA(V), and dimethylarsinous acid (DMA(III)) exposures induce formation of intramitochondrial urothelial inclusions. Arsenite (iAs(III)) also induced intranuclear inclusions in arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice. The arsenic-induced formation of inclusions in the mouse urothelium was dose and time dependent. The inclusions do not occur in iAs-treated rats and do not appear to be related to arsenic-induced urothelial cytotoxicity. Similar inclusions in exfoliated urothelial cells from humans exposed to iAs have been incorrectly identified as micronuclei. We have characterized the urothelial inclusions using transmission electron microscopy (TEM), DNA-specific 4',6-diamidino-2-phenylindole (DAPI), and non-DNA-specific Giemsa staining and determined the arsenical content. The mouse inclusions stained with Giemsa but not with the DAPI stain. Analysis of urothelial mitochondrial- and nuclear-enriched fractions isolated from WT (C57BL/6) and As3mt KO mice exposed to arsenate (iAs(V)) for 4 weeks showed higher levels of iAs(V) in the treated groups. iAs(III) was the major arsenical present in the enriched nuclear fraction from iAs(V)-treated As3mt KO mice. In conclusion, the urothelial cell inclusions induced by arsenicals appear to serve as a detoxifying sequestration mechanism similar to other metals, and they do not represent micronuclei.

Altered lipid partitioning and glucocorticoid availability in CBG-deficient male mice with diet-induced obesity.

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Gulfo, José; Ledda, Angelo; Serra, Elisabet; Cabot, Cristina; Esteve, Montserrat; Grasa, Mar

2016-08-01

To evaluate how deficiency in corticosteroid-binding globulin (CBG), the specific carrier of glucocorticoids, affects glucocorticoid availability and adipose tissue in obesity. C57BL/6 (WT) and CBG-deficient (KO) male mice were fed during 12 weeks with standard or hyperlipidic diet (HL). Glucocorticoid availability and metabolic parameters were assessed. Body weight and food intake were increased in KO compared with WT mice fed a standard diet and were similar when fed a HL diet. Expression of CBG was found in white adipose tissue by immunochemistry, real-time PCR, and Western blot. In obesity, the subcutaneous depot developed less in KO mice compared with WT, which was associated with a minor adipocyte area and peroxisome proliferator-activated receptor-γ expression. Conversely, the epididymal depot displayed higher weight and adipocyte area in KO than in WT mice. CBG deficiency caused a fall of hepatic 11β-hydroxysteroid dehydrogenase type 2 expression and an increase in epidymal adipose tissue, particularly in HL mice. Deficiency in CBG drives lipid partitioning from subcutaneous to visceral adipose depot under a context of lipid excess and differentially modulates 11β-hydroxysteroid dehydrogenase type 2 expression. © 2016 The Obesity Society.

p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

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Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Williams, Stuart [Biomedical Engineering Program, College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Chen, Qin M., E-mail: qchen@email.arizona.edu [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States)

2011-11-15

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients

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Cristiana C. Garcia

2018-05-01

Full Text Available Influenza A virus (IAV infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ, mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single

Genetic markers of a Munc13 protein family member, BAIAP3, are gender specifically associated with anxiety and benzodiazepine abuse in mice and humans.

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Wojcik, Sonja M; Tantra, Martesa; Stepniak, Beata; Man, Kwun-Nok M; Müller-Ribbe, Katja; Begemann, Martin; Ju, Anes; Papiol, Sergi; Ronnenberg, Anja; Gurvich, Artem; Shin, Yong; Augustin, Iris; Brose, Nils; Ehrenreich, Hannelore

2013-07-24

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

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Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Small heterodimer partner (SHP deficiency protects myocardia from lipid accumulation in high fat diet-fed mice.

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Jung Hun Ohn

Full Text Available The small heterodimer partner (SHP regulates fatty acid oxidation and lipogenesis in the liver by regulating peroxisome proliferator-activated receptor (PPAR γ expression. SHP is also abundantly expressed in the myocardium. We investigated the effect of SHP expression on myocardia assessing not only heart structure and function but also lipid metabolism and related gene expression in a SHP deletion animal model. Transcriptional profiling with a microarray revealed that genes participating in cell growth, cytokine signalling, phospholipid metabolism, and extracellular matrix are up-regulated in the myocardia of SHP knockout (KO mice compared to those of wild-type (WT mice (nominal p value < 0.05. Consistent with these gene expression changes, the left ventricular masses of SHP KO mice were significantly higher than WT mice (76.8 ± 20.5 mg vs. 52.8 ± 6.8 mg, P = 0.0093. After 12 weeks of high fat diet (HFD, SHP KO mice gained less weight and exhibited less elevation in serum-free fatty acid and less ectopic lipid accumulation in the myocardium than WT mice. According to microarray analysis, genes regulated by PPARγ1 and PPARα were down-regulated in myocardia of SHP KO mice compared to their expression in WT mice after HFD, suggesting that the reduction in lipid accumulation in the myocardium resulted from a decrease in lipogenesis regulated by PPARγ. We confirmed the reduced expression of PPARγ1 and PPARα target genes such as CD36, medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase by SHP KO after HFD.

IDH2 Deficiency Aggravates Fructose-Induced NAFLD by Modulating Hepatic Fatty Acid Metabolism and Activating Inflammatory Signaling in Female Mice

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Jeong Hoon Pan

2018-05-01

Full Text Available Fructose is a strong risk factor for non-alcoholic fatty liver disease (NAFLD, resulting from the disruption of redox systems by excessive reactive oxygen species production in the liver cells. Of note, recent epidemiological studies indicated that women are more prone to developing metabolic syndrome in response to fructose-sweetened beverages. Hence, we examined whether disruption of the redox system through a deletion of NADPH supplying mitochondrial enzyme, NADP+-dependent isocitrate dehydrogenase (IDH2, exacerbates fructose-induced NAFLD conditions in C57BL/6 female mice. Wild-type (WT and IDH2 knockout (KO mice were treated with either water or 34% fructose water over six weeks. NAFLD phenotypes and key proteins and mRNAs involved in the inflammatory pathway (e.g., NF-κB p65 and IL-1β were assessed. Hepatic lipid accumulation was significantly increased in IDH2 KO mice fed fructose compared to the WT counterpart. Neutrophil infiltration was observed only in IDH2 KO mice fed fructose. Furthermore, phosphorylation of NF-κB p65 and expression of IL-1β was remarkably upregulated in IDH2 KO mice fed fructose, and expression of IκBα was decreased by fructose treatment in both WT and IDH2 KO groups. For the first time, we report our novel findings that IDH2 KO female mice may be more susceptible to fructose-induced NAFLD and the associated inflammatory response, suggesting a mechanistic role of IDH2 in metabolic diseases.

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

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Takemura, Takayo; Yoshida, Yuichi [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Imai, Yasuharu [Department of Gastroenterology, Ikeda Municipal Hospital, Ikeda, Osaka (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University, Graduate School of Medicine and Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center (ProMRes), Ehime University, Shitsukawa, Toon, Ehime (Japan); Iwamoto, Ryo; Mekada, Eisuke [Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Takehara, Tetsuo [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan)

2013-07-26

Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

International Nuclear Information System (INIS)

Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi; Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro; Imai, Yasuharu; Higashiyama, Shigeki; Iwamoto, Ryo; Mekada, Eisuke; Takehara, Tetsuo

2013-01-01

Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis