Ephrinb1 and Ephrinb2 Are Associated with Interleukin-7 Receptor α and Retard Its Internalization from the Cell Surface*

PubMed Central

Luo, Hongyu; Wu, Zenghui; Qi, Shijie; Jin, Wei; Han, Bing; Wu, Jiangping

2011-01-01

IL-7 plays vital roles in thymocyte development, T cell homeostasis, and the survival of these cells. IL-7 receptor α (IL-7Rα) on thymocytes and T cells is rapidly internalized upon IL-7 ligation. Ephrins (Efns) are cell surface molecules and ligands of the largest receptor kinase family, Eph kinases. We discovered that T cell-specific double gene knock-out (dKO) of Efnb1 and Efnb2 in mice led to reduced IL-7Rα expression in thymocytes and T cells, and that IL-7Rα down-regulation was accelerated in dKO CD4 cells upon IL-7 treatment. On the other hand, Efnb1 and Efnb2 overexpression on T cell lymphoma EL4 cells retarded IL-7Rα down-regulation. dKO T cells manifested compromised STAT5 activation and homeostatic proliferation, an IL-7-dependent process. Fluorescence resonance energy transfer and immunoprecipitation demonstrated that Efnb1 and Efnb2 interacted physically with IL-7Rα. Such interaction likely retarded IL-7Rα internalization, as Efnb1 and Efnb2 were not internalized. Therefore, we revealed a novel function of Efnb1 and Efnb2 in stabilizing IL-7Rα expression at the post-translational level, and a previously unknown modus operandi of Efnbs in the regulation of expression of other vital cell surface receptors. PMID:22069310

Antiatherosclerotic and renoprotective effects of ebselen in the diabetic apolipoprotein E/GPx1-double knockout mouse.

PubMed

Chew, Phyllis; Yuen, Derek Y C; Stefanovic, Nada; Pete, Josefa; Coughlan, Melinda T; Jandeleit-Dahm, Karin A; Thomas, Merlin C; Rosenfeldt, Franklin; Cooper, Mark E; de Haan, Judy B

2010-12-01

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress. The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks. Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways. Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

PubMed Central

Estin, Miriam L.; Thompson, Scott B.; Traxinger, Brianna; Fisher, Marlie H.; Friedman, Rachel S.; Jacobelli, Jordan

2017-01-01

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner. PMID:28320969

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca(2+) Channels.

PubMed

Pellegrini, Chiara; Lecci, Sandro; Lüthi, Anita; Astori, Simone

2016-04-01

Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz). Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms. © 2016 Associated Professional Sleep Societies, LLC.

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice*

PubMed Central

Sahu, Bhubanananda; Sun, Wenyu; Perusek, Lindsay; Parmar, Vipulkumar; Le, Yun-Zheng; Griswold, Michael D.; Palczewski, Krzysztof; Maeda, Akiko

2015-01-01

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5−/−Rdh11−/− mice as compared with Rdh5−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision. PMID:26391396

New Advanced Technologies in Stem Cell Therapy

DTIC Science & Technology

2014-11-01

6-8 wks old utrophin/dystrophin double knockout (dKO) mice, a severe animal model of DMD, have an excess of ectopic fat , calcium deposits and...tissues in skeletal muscle alter the tissue environment and induce deregulation of muscle homeostasis; however, the cellular origin of muscle fat ...as a major contributor to ectopic fat cell, calcium deposits and fibrotic tissue formation within dystrophic muscle. In the current study, we propose

Role of NMDA receptor GluN2D subunit in the antidepressant effects of enantiomers of ketamine.

PubMed

Ide, Soichiro; Ikekubo, Yuiko; Mishina, Masayoshi; Hashimoto, Kenji; Ikeda, Kazutaka

2017-11-01

We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA) receptor GluN2D subunit knockout (GluN2D-KO) mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS)-ketamine and (S)-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R)-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R)-ketamine. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

PubMed Central

Kondo, Yoshitaka; Masutomi, Hirofumi; Noda, Yoshihiro; Ozawa, Yusuke; Takahashi, Keita; Handa, Setsuko; Maruyama, Naoki; Shimizu, Takahiko; Ishigami, Akihito

2014-01-01

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion. PMID:25003023

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2

PubMed Central

Ohno, Shouichi; Ikeda, Jun-ichiro; Naito, Yoko; Okuzaki, Daisuke; Sasakura, Towa; Fukushima, Kohshiro; Nishikawa, Yukihiro; Ota, Kaori; Kato, Yorika; Wang, Mian; Torigata, Kosuke; Kasama, Takashi; Uchihashi, Toshihiro; Miura, Daisaku; Yabuta, Norikazu; Morii, Eiichi; Nojima, Hiroshi

2016-01-01

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer. PMID:27982046

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

PubMed

Sasaki, Yuka; Kamiyama, Shuhei; Kamiyama, Azusa; Matsumoto, Konomi; Akatsu, Moe; Nakatani, Yoshihito; Kuwata, Hiroshi; Ishikawa, Yukio; Ishii, Toshiharu; Yokoyama, Chieko; Hara, Shuntaro

2015-11-27

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

Genetic manipulation of the ApoF/Stat2 locus supports an important role for type I interferon signaling in atherosclerosis.

PubMed

Lagor, William R; Fields, David W; Bauer, Robert C; Crawford, Alison; Abt, Michael C; Artis, David; Wherry, E John; Rader, Daniel J

2014-03-01

Apolipoprotein F (ApoF) is a sialoglycoprotein that is a component of the HDL and LDL fractions of human serum. We sought to test the hypothesis that ApoF plays an important role in atherosclerosis in mice by modulating lipoprotein function. Atherosclerosis was assessed in male low density lipoprotein receptor knockout (Ldlr KO) and ApoF/Ldlr double knockout (DKO) mice fed a Western diet for 16 weeks. ApoF/Ldlr DKO mice showed a 39% reduction in lesional area by en face analysis of aortas (p < 0.05), despite no significant differences in plasma lipid parameters. ApoF KO mice had reduced expression of Interferon alpha (IFNα) responsive genes in liver and spleen, as well as impaired macrophage activation. Interferon alpha induced gene 27 like 2a (Ifi27l2a), Oligoadenylate synthetases 2 and 3 (Oas2 and Oas3) were significantly reduced in the ApoF KO mice relative to wild type controls. These effects were attributable to hypomorphic expression of Stat2 in the ApoF KO mice, a critical gene in the Type I IFN pathway that is situated just 425 base pairs downstream of ApoF. These studies implicate STAT2 as a potentially important player in atherosclerosis, and support the growing evidence that the Type I IFN pathway may contribute to this complex disease. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

PubMed Central

Tan, Sih Min; Sharma, Arpeeta; Yuen, Derek Y. C.; Stefanovic, Nada; Krippner, Guy; Mugesh, Govindasamy; Chai, Zhonglin; de Haan, Judy B.

2013-01-01

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation. PMID:23874911

Knockout Mice for Dyslexia Susceptibility Gene Homologs KIAA0319 and KIAA0319L have Unaffected Neuronal Migration but Display Abnormal Auditory Processing

PubMed Central

Guidi, Luiz G; Mattley, Jane; Martinez-Garay, Isabel; Monaco, Anthony P; Linden, Jennifer F; Velayos-Baeza, Antonio

2017-01-01

Abstract Developmental dyslexia is a neurodevelopmental disorder that affects reading ability caused by genetic and non-genetic factors. Amongst the susceptibility genes identified to date, KIAA0319 is a prime candidate. RNA-interference experiments in rats suggested its involvement in cortical migration but we could not confirm these findings in Kiaa0319-mutant mice. Given its homologous gene Kiaa0319L (AU040320) has also been proposed to play a role in neuronal migration, we interrogated whether absence of AU040320 alone or together with KIAA0319 affects migration in the developing brain. Analyses of AU040320 and double Kiaa0319;AU040320 knockouts (dKO) revealed no evidence for impaired cortical lamination, neuronal migration, neurogenesis or other anatomical abnormalities. However, dKO mice displayed an auditory deficit in a behavioral gap-in-noise detection task. In addition, recordings of click-evoked auditory brainstem responses revealed suprathreshold deficits in wave III amplitude in AU040320-KO mice, and more general deficits in dKOs. These findings suggest that absence of AU040320 disrupts firing and/or synchrony of activity in the auditory brainstem, while loss of both proteins might affect both peripheral and central auditory function. Overall, these results stand against the proposed role of KIAA0319 and AU040320 in neuronal migration and outline their relationship with deficits in the auditory system. PMID:29045729

SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule

PubMed Central

Ferdaus, Mohammed Z.; Barber, Karl W.; López‐Cayuqueo, Karen I.; Terker, Andrew S.; Argaiz, Eduardo R.; Gassaway, Brandon M.; Chambrey, Régine; Gamba, Gerardo; Rinehart, Jesse

2016-01-01

Key points STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) phosphorylate and activate the renal Na+–K+–2Cl− cotransporter 2 (NKCC2) and Na+Cl− cotransporter (NCC).Mouse models suggest that OSR1 mainly activates NKCC2‐mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt‐wasting, and generated SPAK/OSR1 double knockout mice to test this.Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase.Compensatory changes in SPAK/OSR1‐independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed.Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K+]. Abstract STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) activate the renal cation cotransporters Na+–K+–2Cl− cotransporter (NKCC2) and Na+–Cl− cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross‐compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt‐wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK‐KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites was lower than in SPAK‐KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K+], with NCC activation serving as the primary effector pathway that modulates K+ secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK‐KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK‐KO and kidney‐specific OSR1 single knockout mice maintained plasma [K+] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K+]. PMID:27068441

Absence of both Sos-1 and Sos-2 in peripheral CD4+ T cells leads to PI3K pathway activation and defects in migration

PubMed Central

Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi; Çuburu, Nicolas; Nguyen, Phan; Sommers, Connie L; Samelson, Lawrence E

2015-01-01

Sos-1 and Sos-2 are ubiquitously expressed Ras-Guanine Exchange Factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4+ T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4+ T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4+ T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration. PMID:25973715

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

Phosphaturic action of fibroblast growth factor 23 in Npt2 null mice.

PubMed

Tomoe, Yuka; Segawa, Hiroko; Shiozawa, Kazuyo; Kaneko, Ichiro; Tominaga, Rieko; Hanabusa, Etsuyo; Aranami, Fumito; Furutani, Junya; Kuwahara, Shoji; Tatsumi, Sawako; Matsumoto, Mitsutu; Ito, Mikiko; Miyamoto, Ken-ichi

2010-06-01

In the present study, we evaluated the roles of type II and type III sodium-dependent P(i) cotransporters in fibroblast growth factor 23 (FGF23) activity by administering a vector encoding FGF23 with the R179Q mutation (FGF23M) to wild-type (WT) mice, Npt2a knockout (KO) mice, Npt2c KO mice, and Npt2a(-/-)Npt2c(-/-) mice (DKO mice). In Npt2a KO mice, FGF23M induced severe hypophosphatemia and markedly decreased the levels of Npt2c, type III Na-dependent P(i) transporter (PiT2) protein, and renal Na/P(i) transport activity. In contrast, in Npt2c KO mice, FGF23M decreased plasma phosphate levels comparable to those in FGF23M-injected WT mice. In DKO mice with severe hypophosphatemia, FGF23M administration did not induce an additional increase in urinary phosphate excretion. FGF23 administration significantly decreased intestinal Npt2b protein levels in WT mice but had no effect in Npt2a, Npt2c, and DKO mice, despite marked suppression of plasma 1,25(OH)(2)D(3) levels in all the mutant mice. The main findings were as follow: 1) FGF23-dependent phosphaturic activity in Npt2a KO mice is dependent on renal Npt2c and PiT-2 protein; 2) in DKO mice, renal P(i) reabsorption is not further decreased by FGF23M, but renal vitamin D synthesis is suppressed; and 3) downregulation of intestinal Npt2b may be mediated by a factor(s) other than 1,25(OH)(2)D(3). These findings suggest that Npt2a, Npt2c, and PiT-2 are necessary for the phosphaturic activity of FGF23. Thus complementary regulation of Npt2 family proteins may be involved in systemic P(i) homeostasis.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo

PubMed Central

Quinn, Stephen J.; Thomsen, Alex R. B.; Pang, Jian L.; Kantham, Lakshmi; Bräuner-Osborne, Hans; Pollak, Martin; Goltzman, David

2013-01-01

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] despite no change in FGF23, suggesting direct regulation of 1,25(OH)2D3 synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range. PMID:23233539

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca2+ Channels

PubMed Central

Pellegrini, Chiara; Lecci, Sandro; Lüthi, Anita; Astori, Simone

2016-01-01

Study Objectives: Low-threshold voltage-gated T-type Ca2+ channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). Methods: We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. Results: CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca2+ currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10–15 Hz), which was accompanied by an increase in the δ band (1–4 Hz). Conclusions: Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms. Citation: Pellegrini C, Lecci S, Lüthi A, Astori S. Suppression of sleep spindle rhythmogenesis in mice with deletion of Cav3.2 and Cav3.3 T-type Ca2+ channels. SLEEP 2016;39(4):875–885. PMID:26612388

Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration.

PubMed

Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi; Çuburu, Nicolas; Nguyen, Phan; Sommers, Connie L; Samelson, Lawrence E

2015-08-01

Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

PubMed

Vilches, Clara; Boiadjieva-Knöpfel, Emilia; Bodoy, Susanna; Camargo, Simone; López de Heredia, Miguel; Prat, Esther; Ormazabal, Aida; Artuch, Rafael; Zorzano, Antonio; Verrey, François; Nunes, Virginia; Palacín, Manuel

2018-04-02

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y + LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo , we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y + LAT1/CD98hc. Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo , and y + LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans. Copyright © 2018 by the American Society of Nephrology.

VAV1-Cre mediated hematopoietic deletion of CBL and CBL-B leads to JMML-like aggressive early-neonatal myeloproliferative disease

PubMed Central

An, Wei; Mohapatra, Bhopal C.; Zutshi, Neha; Bielecki, Timothy A.; Goez, Benjamin T.; Luan, Haitao; Iseka, Fany; Mushtaq, Insha; Storck, Matthew D.; Band, Vimla; Band, Hamid

2016-01-01

CBL and CBL-B ubiquitin ligases play key roles in hematopoietic stem cell homeostasis and their aberrations are linked to leukemogenesis. Mutations of CBL, often genetically-inherited, are particularly common in Juvenile Myelomonocytic Leukemia (JMML), a disease that manifests early in children. JMML is fatal unless corrected by bone marrow transplant, which is effective in only half of the recipients, stressing the need for animal models that recapitulate the key clinical features of this disease. However, mouse models established so far only develop hematological malignancy in adult animals. Here, using VAV1-Cre-induced conditional CBL/CBL-B double knockout (DKO) in mice, we established an animal model that exhibits a neonatal myeloproliferative disease (MPD). VAV1-Cre induced DKO mice developed a strong hematological phenotype at postnatal day 10, including severe leukocytosis and hepatomegaly, bone marrow cell hypersensitivity to cytokines including GM-CSF, and rapidly-progressive disease and invariable lethality. Interestingly, leukemic stem cells were most highly enriched in neonatal liver rather than bone marrow, which, along with the spleen and thymus, were hypo-cellular. Nonetheless, transplantation assays showed that both DKO bone marrow and liver cells can initiate leukemic disease in the recipient mice with seeding of both spleen and bone marrow. Together, our results support the usefulness of the new hematopoietic-specific CBL/CBL-B double KO animal model to study JMML-related pathogenesis and to further understand the function of CBL family proteins in regulating fetal and neonatal hematopoiesis. To our knowledge, this is the first mouse model that exhibits neonatal MPD in infancy, by day 10 of postnatal life. PMID:27449297

The non-diuretic hypotensive effects of thiazides are enhanced during volume depletion states

PubMed Central

Alshahrani, Saeed; Rapoport, Robert M.; Zahedi, Kamyar; Jiang, Min; Nieman, Michelle; Barone, Sharon; Meredith, Andrea L.; Lorenz, John N.; Rubinstein, Jack

2017-01-01

Thiazide derivatives including Hydrochlorothiazide (HCTZ) represent the most common treatment of mild to moderate hypertension. Thiazides initially enhance diuresis via inhibition of the kidney Na+-Cl- Cotransporter (NCC). However, chronic volume depletion and diuresis are minimal while lowered blood pressure (BP) is maintained on thiazides. Thus, a vasodilator action of thiazides is proposed, likely via Ca2+-activated K+ (BK) channels in vascular smooth muscles. This study ascertains the role of volume depletion induced by salt restriction or salt wasting in NCC KO mice on the non-diuretic hypotensive action of HCTZ. HCTZ (20mg/kg s.c.) lowered BP in 1) NCC KO on a salt restricted diet but not with normal diet; 2) in volume depleted but not in volume resuscitated pendrin/NCC dKO mice; the BP reduction occurs without any enhancement in salt excretion or reduction in cardiac output. HCTZ still lowered BP following treatment of NCC KO on salt restricted diet with paxilline (8 mg/kg, i.p.), a BK channel blocker, and in BK KO and BK/NCC dKO mice on salt restricted diet. In aortic rings from NCC KO mice on normal and low salt diet, HCTZ did not alter and minimally decreased maximal phenylephrine contraction, respectively, while contractile sensitivity remained unchanged. These results demonstrate 1) the non-diuretic hypotensive effects of thiazides are augmented with volume depletion and 2) that the BP reduction is likely the result of HCTZ inhibition of vasoconstriction through a pathway dependent on factors present in vivo, is unrelated to BK channel activation, and involves processes associated with intravascular volume depletion. PMID:28719636

EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

PubMed

Cheng, Catherine; Fowler, Velia M; Gong, Xiaohua

2017-09-01

Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results indicate that EphA2 and ephrin-A5 do not form a lens receptor-ligand pair, and that EphA2 and ephrin-A5 have other binding partners in the lens to help align differentiating equatorial epithelial cells or maintain the anterior epithelium, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

New Advanced Technologies in Stem Cell Therapy

DTIC Science & Technology

2013-09-01

infiltration in skeletal muscle. Ectopic fat accumulation in skeletal muscle can be seen not only in myopathies but also in several disorders...mice; however, the source of the ectopic fat tissue within the skeletal muscle is unknown. In this study, we provide evidence that the RACs, PDGFRα...mesenchymal progenitor cells, are responsible for increased fat cell formation in the skeletal muscle of dKO mice. We observed that dKO-RACs had

Matrix metalloproteinase-9 deletion rescues auditory evoked potential habituation deficit in a mouse model of Fragile X Syndrome

PubMed Central

Lovelace, Jonathan W.; Wen, Teresa H.; Reinhard, Sarah; Hsu, Mike S.; Sidhu, Harpreet; Ethell, Iryna M.; Binder, Devin K.; Razak, Khaleel A.

2016-01-01

Sensory processing deficits are common in autism spectrum disorders, but the underlying mechanisms are unclear. Fragile X Syndrome (FXS) is a leading genetic cause of intellectual disability and autism. Electrophysiological responses in humans with FXS show reduced habituation with sound repetition and this deficit may underlie auditory hypersensitivity in FXS. Our previous study in Fmr1 knockout (KO) mice revealed an unusually long state of increased sound-driven excitability in auditory cortical neurons suggesting that cortical responses to repeated sounds may exhibit abnormal habituation as in humans with FXS. Here, we tested this prediction by comparing cortical event related potentials (ERP) recorded from wildtype (WT) and Fmr1 KO mice. We report a repetition-rate dependent reduction in habituation of N1 amplitude in Fmr1 KO mice and show that matrix metalloproteinase −9 (MMP-9), one of the known FMRP targets, contributes to the reduced ERP habituation. Our studies demonstrate a significant up-regulation of MMP-9 levels in the auditory cortex of adult Fmr1 KO mice, whereas a genetic deletion of Mmp-9 reverses ERP habituation deficits in Fmr1 KO mice. Although the N1 amplitude of Mmp-9/Fmr1 DKO recordings was larger than WT and KO recordings, the habituation of ERPs in Mmp-9/Fmr1 DKO mice is similar to WT mice implicating MMP-9 as a potential target for reversing sensory processing deficits in FXS. Together these data establish ERP habituation as a translation relevant, physiological pre-clinical marker of auditory processing deficits in FXS and suggest that abnormal MMP-9 regulation is a mechanism underlying auditory hypersensitivity in FXS. PMID:26850918

Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.

PubMed

Parks, Scott K; Cormerais, Yann; Durivault, Jerome; Pouyssegur, Jacques

2017-02-07

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.

Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells

PubMed Central

Parks, Scott K.; Cormerais, Yann; Durivault, Jerome; Pouyssegur, Jacques

2017-01-01

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy. PMID:28055960

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice.

PubMed

Wu, Pu; Shen, Qian; Dong, Suzhen; Xu, Zhiliang; Tsien, Joe Z; Hu, Yinghe

2008-10-01

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Abrogated Freud-1/CC2D1A repression of 5-HT1A autoreceptors induces fluoxetine-resistant anxiety/depression-like behavior

PubMed Central

Vahid-Ansari, Faranak; Daigle, Mireille; Manzini, M. Chiara; Tanaka, Kenji F.; Hen, René; Geddes, Sean D.; Béïque, Jean-Claude; James, Jonathan; Merali, Zul; Albert, Paul R.

2017-01-01

Freud-1/CC2D1A represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knockout of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knockout (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behaviour was seen upon knockout of Freud-1 on the 5-HT1A autoreceptor-negative background, rather a reduction in depression-like behaviour emerged. These studies implicate transcriptional dys-regulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors like Freud-1 to restore transcriptional balance may augment response to antidepressant treatment. PMID:29101244

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

PubMed

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.

Acute Heat-Evoked Temperature Sensation Is Impaired but Not Abolished in Mice Lacking TRPV1 and TRPV3 Channels

PubMed Central

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. PMID:24925072

Prostaglandin-E2 Mediated Increase in Calcium and Phosphate Excretion in a Mouse Model of Distal Nephron Salt Wasting

PubMed Central

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Alshahrani, Saeed; Brooks, Marybeth; McCormack, Francis X.; Smith, Roger D.; Zahedi, Kamyar

2016-01-01

Contribution of salt wasting and volume depletion to the pathogenesis of hypercalciuria and hyperphosphaturia is poorly understood. Pendrin/NCC double KO (pendrin/NCC-dKO) mice display severe salt wasting under basal conditions and develop profound volume depletion, prerenal renal failure, and metabolic alkalosis and are growth retarded. Microscopic examination of the kidneys of pendrin/NCC-dKO mice revealed the presence of calcium phosphate deposits in the medullary collecting ducts, along with increased urinary calcium and phosphate excretion. Confirmatory studies revealed decreases in the expression levels of sodium phosphate transporter-2 isoforms a and c, increases in the expression of cytochrome p450 family 4a isotypes 12 a and b, as well as prostaglandin E synthase 1, and cyclooxygenases 1 and 2. Pendrin/NCC-dKO animals also had a significant increase in urinary prostaglandin E2 (PGE-2) and renal content of 20-hydroxyeicosatetraenoic acid (20-HETE) levels. Pendrin/NCC-dKO animals exhibit reduced expression levels of the sodium/potassium/2chloride co-transporter 2 (NKCC2) in their medullary thick ascending limb. Further assessment of the renal expression of NKCC2 isoforms by quantitative real time PCR (qRT-PCR) reveled that compared to WT mice, the expression of NKCC2 isotype F was significantly reduced in pendrin/NCC-dKO mice. Provision of a high salt diet to rectify volume depletion or inhibition of PGE-2 synthesis by indomethacin, but not inhibition of 20-HETE generation by HET0016, significantly improved hypercalciuria and salt wasting in pendrin/NCC dKO mice. Both high salt diet and indomethacin treatment also corrected the alterations in NKCC2 isotype expression in pendrin/NCC-dKO mice. We propose that severe salt wasting and volume depletion, irrespective of the primary originating nephron segment, can secondarily impair the reabsorption of salt and calcium in the thick ascending limb of Henle and/or proximal tubule, and reabsorption of sodium and phosphate in the proximal tubule via processes that are mediated by PGE-2. PMID:27442254

Environment enrichment rescues the neurodegenerative phenotypes in presenilins-deficient mice.

PubMed

Dong, Suzhen; Li, Chunxia; Wu, Pu; Tsien, Joe Z; Hu, Yinghe

2007-07-01

Presenilin (PS) 1 and 2 conditional double knockout (cDKO) mice show progressive memory dysfunction and forebrain degeneration. Gene expression profiling results revealed a strong activation of immunity and inflammation responses in the brains of 10-month-old cDKO mice. As environmental enrichment (EE) has been shown to be able to improve memory and induce neurogenesis of the brain, we assessed the effects of EE on the memory performance and the neurodegeneration in cDKO mice. We found that EE effectively enhanced memory and partially rescued the forebrain atrophy of the cDKO mice. Our results suggest that immunity and inflammation could play important roles in the neurodegeneration of cDKO mice. Furthermore, the beneficial effects of EE may be associated with the inhibition of the expression of immunity and inflammation-related genes in the brain.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

Matrix metalloproteinase-9 deletion rescues auditory evoked potential habituation deficit in a mouse model of Fragile X Syndrome.

PubMed

Lovelace, Jonathan W; Wen, Teresa H; Reinhard, Sarah; Hsu, Mike S; Sidhu, Harpreet; Ethell, Iryna M; Binder, Devin K; Razak, Khaleel A

2016-05-01

Sensory processing deficits are common in autism spectrum disorders, but the underlying mechanisms are unclear. Fragile X Syndrome (FXS) is a leading genetic cause of intellectual disability and autism. Electrophysiological responses in humans with FXS show reduced habituation with sound repetition and this deficit may underlie auditory hypersensitivity in FXS. Our previous study in Fmr1 knockout (KO) mice revealed an unusually long state of increased sound-driven excitability in auditory cortical neurons suggesting that cortical responses to repeated sounds may exhibit abnormal habituation as in humans with FXS. Here, we tested this prediction by comparing cortical event related potentials (ERP) recorded from wildtype (WT) and Fmr1 KO mice. We report a repetition-rate dependent reduction in habituation of N1 amplitude in Fmr1 KO mice and show that matrix metalloproteinase-9 (MMP-9), one of the known FMRP targets, contributes to the reduced ERP habituation. Our studies demonstrate a significant up-regulation of MMP-9 levels in the auditory cortex of adult Fmr1 KO mice, whereas a genetic deletion of Mmp-9 reverses ERP habituation deficits in Fmr1 KO mice. Although the N1 amplitude of Mmp-9/Fmr1 DKO recordings was larger than WT and KO recordings, the habituation of ERPs in Mmp-9/Fmr1 DKO mice is similar to WT mice implicating MMP-9 as a potential target for reversing sensory processing deficits in FXS. Together these data establish ERP habituation as a translation relevant, physiological pre-clinical marker of auditory processing deficits in FXS and suggest that abnormal MMP-9 regulation is a mechanism underlying auditory hypersensitivity in FXS. Fragile X Syndrome (FXS) is the leading known genetic cause of autism spectrum disorders. Individuals with FXS show symptoms of auditory hypersensitivity. These symptoms may arise due to sustained neural responses to repeated sounds, but the underlying mechanisms remain unclear. For the first time, this study shows deficits in habituation of neural responses to repeated sounds in the Fmr1 KO mice as seen in humans with FXS. We also report an abnormally high level of matrix metalloprotease-9 (MMP-9) in the auditory cortex of Fmr1 KO mice and that deletion of Mmp-9 from Fmr1 KO mice reverses habituation deficits. These data provide a translation relevant electrophysiological biomarker for sensory deficits in FXS and implicate MMP-9 as a target for drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

Ibuprofen partially attenuates neurodegenerative symptoms in presenilin conditional double-knockout mice.

PubMed

Dong, Z; Yan, L; Huang, G; Zhang, L; Mei, B; Meng, B

2014-06-13

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug that reportedly reduces the risk of Alzheimer's disease (AD) development. The anti-inflammatory effect of ibuprofen occurred via inhibition of cyclooxygenases and anti-amyloidogenesis through modulation of γ-secretase. Presenilin 1 and 2 conditional double-knockout (cDKO) mice exhibited age-dependent memory impairment and forebrain degeneration without elevation of amyloid β deposition. Therefore, cDKO mice can be an ideal animal model on which to independently test the effects of ibuprofen anti-inflammatory properties on the prevention of AD. Three- and six-month-old cDKO mice were fed diet containing 375 ppm ibuprofen for six months. After multiple, well-validated behavioral tests, treatment with ibuprofen improved cognition-related behavioral performance, and drug efficacy was correlated with the timing of administration. Ibuprofen was more effective on six-month-old than on three-month-old cDKO mice. Biochemical analysis demonstrated that the effects of ibuprofen on glial fibrillary acidic protein and CD68 expression levels were uneven in different brain regions of cDKO mice and that age also influenced such effects. Tau hyperphosphorylation and the cleavage of caspase-3 decreased after ibuprofen treatment, and this effect was more significant in the older than the younger group of mice, which was consistent with the results of behavioral tests. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

The Role of the New mTOR Complex, mTORC2, in Autism Spectrum Disorders

DTIC Science & Technology

2016-10-01

memory in Pten fb-KO mice. a, Schematic of experimental design . b, For contextual fear conditioning, freezing times were recorded 24 hr after...official Department of the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No...fb-DKO mice, the opposite is true , namely mTORC2 activity remains up-regulated by mTORC1 activity is normalized (Fig. 1). Hence, conditional

Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells.

PubMed

Fujimura, Tatsuya; Takahagi, Yoichi; Shigehisa, Tamotsu; Nagashima, Hiroshi; Miyagawa, Shuji; Shirakura, Ryota; Murakami, Hiroshi

2008-09-01

The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.

Attenuation of neurodegenerative phenotypes in Alzheimer-like presenilin 1/presenilin 2 conditional double knockout mice by EUK1001, a promising derivative of xanomeline.

PubMed

Wang, Dong; Yang, Liguo; Su, Jingjing; Niu, Yan; Lei, Xiaoping; Xiong, Juan; Cao, Xiaohua; Hu, Yinghe; Mei, Bing; Hu, Jin-Feng

2011-07-01

The M1/M4-preferring muscarinic agonist xanomeline was found to have some benefit in the treatment of the memory impairment of Alzheimer's disease (AD), but side effects precluded further development. EUK1001, a fluorinated derivative of xanomeline, because of greater affinity for M1 muscarinic receptors, is likely to have a significantly better side effect profile than xanomeline. We have now studied the effects of 3-month chronic administration of EUK1001 and xanomeline (0.5mg/kg/day) in AD-like presenilin 1/presenilin 2 conditional double knockout (PS cDKO) mice. Only EUK1001 was found to significantly ameliorate the deficit in recognition memory. Histological analysis demonstrated partial attenuation of the brain atrophy in EUK1001-treated PS cDKO mice and minimal effect in the xanomeline-treated mice. Both compounds effectively suppressed the elevation of brain tau phosphorylation in the PS cDKO mice, but neither inhibited the increased inflammatory responses. These results indicate that EUK1001 showed superiority to xanomeline with regard to attenuation of several AD-like neurodegenerative phenotypes in PS cDKO mice. These results suggest further investigation of the development of EUK1001 for the treatment of AD is indicated. Copyright © 2011 Elsevier Inc. All rights reserved.

Chemical and genetic validation of dihydrofolate reductase–thymidylate synthase as a drug target in African trypanosomes

PubMed Central

Sienkiewicz, Natasha; Jarosławski, Szymon; Wyllie, Susan; Fairlamb, Alan H

2008-01-01

The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase–thymidylate synthase (DHFR–TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR–TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR–TS is essential for parasite survival and represents a promising target for drug discovery. PMID:18557814

Metameric pattern of intervertebral disc/vertebral body is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somites in the mouse embryo.

PubMed

Takahashi, Yu; Yasuhiko, Yukuto; Takahashi, Jun; Takada, Shinji; Johnson, Randy L; Saga, Yumiko; Kanno, Jun

2013-08-15

The vertebrae are derived from the sclerotome of somites. Formation of the vertebral body involves a process called resegmentation, by which the caudal half of a sclerotome is combined with the rostral half of the next sclerotome. To elucidate the relationship between resegmentation and rostro-caudal patterning of somite, we used the Uncx4.1-LacZ transgene to characterize the resegmentation process. Our observations suggested that in the thoracic and lumbar vertebrae, the Uncx4.1-expressing caudal sclerotome gave rise to the intervertebral disc (IVD) and rostral portion of the vertebral body (VB). In the cervical vertebrae, the Uncx4.1-expressing caudal sclerotome appeared to contribute to the IVD and both caudal and rostral ends of the VB. This finding suggests that the rostro-caudal gene expression boundary does not necessarily coincide with the resegmentation boundary. This conclusion was supported by analyses of Mesp2 KO and Ripply1/2 double KO embryos lacking rostral and caudal properties, respectively. Resegmentation was not observed in Mesp2 KO embryos, but both the IVD and whole VB were formed from the caudalized sclerotome. Expression analysis of IVD marker genes including Pax1 in the wild-type, Mesp2 KO, and Ripply1/2 DKO embryos also supported the idea that a metameric pattern of IVD/VB is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somite. However, in the lumbar region, IVD differentiation appeared to be stimulated by the caudal property and suppressed by the rostral property. Therefore, we propose that rostro-caudal patterning of somites is not a prerequisite for metameric patterning of the IVD and VB, but instead required to stimulate IVD differentiation in the caudal half of the sclerotome. Copyright © 2013 Elsevier Inc. All rights reserved.

Muscle Structure Influences Utrophin Expression in mdx Mice

PubMed Central

Banks, Glen B.; Combs, Ariana C.; Odom, Guy L.; Bloch, Robert J.; Chamberlain, Jeffrey S.

2014-01-01

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin−/−, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD. PMID:24922526

Knocking out P2X receptors reduces transmitter secretion in taste buds.

PubMed

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

2011-09-21

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.

Sodium–hydrogen exchanger NHA1 and NHA2 control sperm motility and male fertility

PubMed Central

Chen, Su-Ren; Chen, M; Deng, S-L; Hao, X-X; Wang, X-X; Liu, Y-X

2016-01-01

Our previous work identified NHA1, a testis-specific sodium–hydrogen exchanger, is specifically localized on the principal piece of mouse sperm flagellum. Our subsequent study suggested that the number of newborns and fertility rate of NHA1-vaccinated female mice are significantly stepped down. In order to define the physiological function of NHA1 in spermatozoa, we generated Nha1Fx/Fx, Zp3-Cre (hereafter called Nha1 cKO) mice and found that Nha1 cKO males were viable and subfertile with reduced sperm motility. Notably, cyclic AMP (cAMP) synthesis by soluble adenylyl cyclase (sAC) was attenuated in Nha1 cKO spermatozoa and cAMP analogs restored sperm motility. Similar to Nha1 cKO males, Nha2Fx/Fx, Zp3-Cre (hereafter called Nha2 cKO) male mice were subfertile, indicating these two Nha genes may be functionally redundant. Furthermore, we demonstrated that male mice lacking Nha1 and Nha2 genes (hereafter called Nha1/2 dKO mice) were completely infertile, with severely diminished sperm motility owing to attenuated sAC-cAMP signaling. Importantly, principal piece distribution of NHA1 in spermatozoa are phylogenetically conserved in spermatogenesis. Collectively, our data revealed that NHA1 and NHA2 function as a key sodium–hydrogen exchanger responsible for sperm motility after leaving the cauda epididymidis. PMID:27010853

Dnmt1 and Dnmt3a are required for the maintenance of DNA methylation and synaptic function in adult forebrain neurons

PubMed Central

Feng, Jian; Zhou, Yu; Campbell, Susan L.; Le, Thuc; Li, En; Sweatt, J. David; Silva, Alcino J.; Fan, Guoping

2011-01-01

Dnmt1 and Dnmt3a, two major DNA methyltransferases, are expressed in postmitotic neurons, but their function in the central nervous system (CNS) is unclear. We generated conditional mutant mice that lack either Dnmt1, or Dnmt3a, or both exclusively in forebrain excitatory neurons and found only double knockout (DKO) mice exhibited abnormal hippocampal CA1 long-term plasticity and deficits of learning and memory. While no neuronal loss was found, the size of hippocampal neurons in DKO was smaller; furthermore, DKO neurons showed a deregulation of gene expression including class I MHC and Stat1 that are known to play a role in synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons. PMID:20228804

Regulation of Effector Treg Cells in Murine Lupus.

PubMed

Chandrasekaran, Uma; Yi, Woelsung; Gupta, Sanjay; Weng, Chien-Huan; Giannopoulou, Eugenia; Chinenov, Yurii; Jessberger, Rolf; Weaver, Casey T; Bhagat, Govind; Pernis, Alessandra B

2016-06-01

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development. © 2016, American College of Rheumatology.

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

PubMed Central

Dykes, Iain M.; Tempest, Lynne; Lee, Su-In; Turner, Eric E.

2011-01-01

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal (TG) and dorsal root ganglia (DRG) co-express the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knockout mice (DKO mice). Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knockout alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a -regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes. PMID:21734270

Ocular inflammation in HLA-B27 transgenic mice reveals a potential role for MHC class I in corneal immune privilege.

PubMed

Lin, Aifeng; Guo, Xiaoxin; Inman, Robert D; Sivak, Jeremy M

2015-01-01

HLA-B27 is a major histocompatibility complex class I (MHCI) allele that has been closely associated with the development of ankylosing spondylitis and acute anterior uveitis (AAU), the most common form of uveitis worldwide. We have been characterizing the phenotypes of transgenic mice carrying a human HLA-B27 allele, but that are deficient in endogenous mouse MHCI genes (H-2K(-/-) and H-2D(-/-) double knockout, or DKO) to create the HLA-B27/DKO line. In maintaining and expanding this colony, we observed a rare sporadic severe central keratitis that developed in transgenic animals, but that was not present in wild-type (WT) animals. The corneas of affected HLA-B27/DKO and DKO mice were compared to their WT counterparts by staining with standard histological methods for markers of inflammation and neovascularization. A model of experimental corneal inflammation was subsequently used to test the responses of each genotype to insult. We identified a previously unreported corneal pathology in naïve HLA-B27/DKO mice, and we describe significantly prolonged CD4(+)- and CD8(+)-associated inflammation in these animals following an experimentally induced corneal injury. These results demonstrate an increased T-cell response in B27/DKO corneas due to the expression of the HLA-B27 allele, suggesting that low MHCI expression in WT corneas is an important contributor to immune privilege.

Knocking out P2X receptors reduces transmitter secretion in taste buds

PubMed Central

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

2011-01-01

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

Xenobiotic Nuclear Receptor Signaling Determines Molecular Pathogenesis of Progressive Familial Intrahepatic Cholestasis.

PubMed

Kim, Kang Ho; Choi, Jong Min; Li, Feng; Arizpe, Armando; Wooton-Kee, Clavia Ruth; Anakk, Sayeepriyadarshini; Jung, Sung Yun; Finegold, Milton J; Moore, David D

2018-06-01

Progressive familial intrahepatic cholestasis (PFIC) is a genetically heterogeneous disorder of bile flow disruption due to abnormal canalicular transport or impaired bile acid (BA) metabolism, causing excess BA accumulation and liver failure. We previously reported an intrahepatic cholestasis mouse model based on loss of function of both farnesoid X receptor (FXR; NR1H4) and a small heterodimer partner (SHP; NR0B2) [double knockout (DKO)], which has strong similarities to human PFIC5. We compared the pathogenesis of DKO livers with that of another intrahepatic cholestasis model, Bsep-/-, which represents human PFIC2. Both models exhibit severe hepatomegaly and hepatic BA accumulation, but DKO showed greater circulating BA and liver injury, and Bsep-/- had milder phenotypes. Molecular profiling of BAs uncovered specific enrichment of cholic acid (CA)-derived BAs in DKO livers but chenodeoxycholate-derived BAs in Bsep-/- livers. Transcriptomic and proteomic analysis revealed specific activation of CA synthesis and alternative basolateral BA transport in DKO but increased chenodeoxycholic acid synthesis and canalicular transport in Bsep-/-. The constitutive androstane receptor (CAR)/pregnane X receptor (PXR)-CYP2B/CYP2C axis is activated in DKO livers but not in other cholestasis models. Loss of this axis in Fxr:Shp:Car:Pxr quadruple knockouts blocked Cyp2b/Cyp2c gene induction, impaired bilirubin conjugation/elimination, and increased liver injury. Differential CYP2B expression in DKO and Bsep-/- was recapitulated in human PFIC5 and PFIC2 livers. In conclusion, loss of FXR/SHP results in distinct molecular pathogenesis and CAR/PXR activation, which promotes Cyp2b/Cyp2c gene transcription and bilirubin clearance. CAR/PXR activation was not observed in Bsep-/- mice or PFIC2 patients. These findings provide a deeper understanding of the heterogeneity of intrahepatic cholestasis.

Compromised renal microvascular reactivity of angiotensin type 1 double null mice.

PubMed

Park, Sungmi; Bivona, Benjamin J; Harrison-Bernard, Lisa M

2007-07-01

Angiotensin type 1A (AT(1A)) and 1B (AT(1B)) receptor deletion (AT1DKO) results in renal microvascular disease, tubulointerstitial injury, and reduced blood pressure. To test the hypothesis that renal preglomerular responses to angiotensin (ANG) II are mediated by AT(1A) and AT(1B) receptors, experiments were performed in AT1DKO mice using the in vitro blood perfused juxtamedullary nephron technique. Kidneys were harvested from AT1DKO and wild-type (WT) mice and bathed with ANG II (1-100 nM), norepinephrine (NE; 100-1,000 nM), or acetylcholine (ACh; 10 microM). Baseline diameters of afferent (19.5 +/- 0.7 and 13.9 +/- 0.7 microm, n = 17 and 16) and efferent (15.5 +/- 2.1 and 10.8 +/- 1.0 microm, n = 4 and 7) arterioles of AT1DKO were significantly larger than WT. Afferent and efferent arteriolar responses to ANG II, 100, and 300 nM NE were absent in AT1DKO; although significant constriction to 1 microM NE was observed (-17 +/- 5 and -23 +/- 6%, respectively). Afferent arterioles of WT mice dilated significantly in response to ACh (15.1 +/- 0.6 to 17.0 +/- 1.2 microm, n = 6); however, arterioles from AT1DKO tended to contract (19.9 +/- 1.2 to 17.8 +/- 1.6 microm; n = 6, P = 0.06). In summary, loss of ANG II-induced contraction, reduced vasoconstriction to NE, and endothelial cell dysfunction contribute to the renal vascular phenotype of AT1DKO mice. We conclude that ANG II signaling via the AT(1) receptor plays a pivotal role in basal renal microvascular tone and effectiveness to respond to vasoconstrictor and vasodilator agonists.

The Ovary Is an Alternative Site of Origin for High-Grade Serous Ovarian Cancer in Mice

PubMed Central

Coffey, Donna M.; Ma, Lang; Matzuk, Martin M.

2015-01-01

Although named “ovarian cancer,” it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53R172H, into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53R172H-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice. PMID:25815421

The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

PubMed

Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

2015-06-01

Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Disruption of Genes Encoding eIF4E Binding Proteins-1 And -2 Does Not Alter Basal or Sepsis-Induced Changes in Skeletal Muscle Protein Synthesis in Male or Female Mice

PubMed Central

Steiner, Jennifer L.; Pruznak, Anne M.; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R.; Lang, Charles H.

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding. PMID:24945486

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

PubMed

Steiner, Jennifer L; Pruznak, Anne M; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R; Lang, Charles H

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit calcineurin-induced myocardial hypertrophy.

PubMed

Kreusser, Michael M; Lehmann, Lorenz H; Keranov, Stanislav; Hoting, Marc-Oscar; Oehl, Ulrike; Kohlhaas, Michael; Reil, Jan-Christian; Neumann, Kay; Schneider, Michael D; Hill, Joseph A; Dobrev, Dobromir; Maack, Christoph; Maier, Lars S; Gröne, Hermann-Josef; Katus, Hugo A; Olson, Eric N; Backs, Johannes

2014-10-07

Ca(2+)-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca(2+) handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. We established a mouse model in which CaMKII's activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure. © 2014 American Heart Association, Inc.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

PubMed

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

2016-11-01

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Mice lacking GRIP1/2 show increased social interactions and enhanced phosphorylation at GluA2-S880.

PubMed

Han, Mei; Mejias, Rebeca; Chiu, Shu-Ling; Rose, Rebecca; Adamczyk, Abby; Huganir, Richard; Wang, Tao

2017-03-15

Glutamate receptor interacting proteins 1 and 2 (GRIP1/2) play an important role in regulating synaptic trafficking of AMPA receptor 2/3 (GluA2/3) and synaptic strength. Gain-of-function GRIP1 mutations are implicated in social behavioral deficits in autism. To study mechanisms of Grip1/2-mediated AMPA signaling in the regulation of social behaviors, we performed social behavioral testing on neuron-specific Grip1/2-double knockout (DKO) and wild type (WT) mice that are matched for age, sex, and strain background. We determined the expression profile of key signaling proteins in AMPAR, mGluR, mTOR, and GABA pathways in frontal cortex, striatum, and cerebellum of DKO mice. Compared to WT mice, DKO mice show increased sociability in a modified three-chamber social behavioral test [mean±sem for interaction time in seconds; WT: 44.0±5.0; n=10; DKO: 81.0±9.0; n=9; two factor repeated measures ANOVA: F(1,37)=14.45; p<0.01 and planned t-test; p<0.01] and in a dyadic male-male social interaction test (mean±sem for total time in seconds: sniffing, WT-WT, 18.9±1.1; WT-DKO, 42.5±2.1; t-test: p<0.001; following, WT-WT, 7.7±0.72; WT-DKO,14.4±1.8; t-test: p<0.001). Immunoblot studies identified an increase in phosphorylation at GluA2-Serine 880 (GluA2-pS880) in frontal cortex (mean±sem; WT: 0.69±0.06, n=5; DKO: 0.96±0.06, n=6; t-test; p<0.05) and reduced GABAβ3 expression in striatum (mean±sem; WT: 1.16±0.04, n=4; DKO: 0.95±0.06, n=4; t-test; p<0.05) in DKO mice. GluA2-S880 phosphorylation is known to regulate GluA2synaptic recycling, AMPA signaling strength and plasticity. GABAβ3 has been implicated in the etiology and pathogenesis in autism. These data support an important role of Grip1/2-mediated AMPA signaling in regulating social behaviors and disturbance of glutamate- and GABA-signaling in specialized brain regions in autism-related social behavioral deficits. Copyright © 2017 Elsevier B.V. All rights reserved.

The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

PubMed

Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

2014-06-06

The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

Degenerative phenotypes caused by the combined deficiency of murine HIP1 and HIP1r are rescued by human HIP1.

PubMed

Bradley, Sarah V; Hyun, Teresa S; Oravecz-Wilson, Katherine I; Li, Lina; Waldorff, Erik I; Ermilov, Alexander N; Goldstein, Steven A; Zhang, Claire X; Drubin, David G; Varela, Kate; Parlow, Al; Dlugosz, Andrzej A; Ross, Theodora S

2007-06-01

The members of the huntingtin-interacting protein-1 (HIP1) family, HIP1 and HIP1-related (HIP1r), are multi-domain proteins that interact with inositol lipids, clathrin and actin. HIP1 is over-expressed in a variety of cancers and both HIP1 and HIP1r prolong the half-life of multiple growth factor receptors. To better understand the physiological importance of the HIP1 family in vivo, we have analyzed a large cohort of double Hip1/Hip1r knockout (DKO) mice. All DKO mice were dwarfed, afflicted with severe vertebral defects and died in early adulthood. These phenotypes were not observed during early adulthood in the single Hip1 or Hip1r knockouts, indicating that HIP1 and HIP1r compensate for one another. Despite the ability of HIP1 and HIP1r to modulate growth factor receptor levels when over-expressed, studies herein using DKO fibroblasts indicate that the HIP1 family is not necessary for endocytosis but is necessary for the maintenance of diverse adult tissues in vivo. To test if human HIP1 can function similar to mouse HIP1, transgenic mice with 'ubiquitous' expression of the human HIP1 cDNA were generated and crossed with DKO mice. Strikingly, the compound human HIP1 transgenic DKO mice were completely free from dwarfism and spinal defects. This successful rescue demonstrates that the human HIP1 protein shares some interchangeable functions with both HIP1 and HIP1r in vivo. In addition, we conclude that the degenerative phenotypes seen in the DKO mice are due mainly to HIP1 and HIP1r protein deficiency rather than altered expression of neighboring genes or disrupted intronic elements.

Disruption of both nesprin 1 and desmin results in nuclear anchorage defects and fibrosis in skeletal muscle

PubMed Central

Chapman, Mark A.; Zhang, Jianlin; Banerjee, Indroneal; Guo, Ling T.; Zhang, Zhiwei; Shelton, G. Diane; Ouyang, Kunfu; Lieber, Richard L.; Chen, Ju

2014-01-01

Proper localization and anchorage of nuclei within skeletal muscle is critical for cellular function. Alterations in nuclear anchoring proteins modify a number of cellular functions including mechanotransduction, nuclear localization, chromatin positioning/compaction and overall organ function. In skeletal muscle, nesprin 1 and desmin are thought to link the nucleus to the cytoskeletal network. Thus, we hypothesize that both of these factors play a key role in skeletal muscle function. To examine this question, we utilized global ablation murine models of nesprin 1, desmin or both nesprin 1 and desmin. Herein, we have created the nesprin-desmin double-knockout (DKO) mouse, eliminating a major fraction of nuclear-cytoskeletal connections and enabling understanding of the importance of nuclear anchorage in skeletal muscle. Globally, DKO mice are marked by decreased lifespan, body weight and muscle strength. With regard to skeletal muscle, DKO myonuclear anchorage was dramatically decreased compared with wild-type, nesprin 1−/− and desmin−/− mice. Additionally, nuclear-cytoskeletal strain transmission was decreased in DKO skeletal muscle. Finally, loss of nuclear anchorage in DKO mice coincided with a fibrotic response as indicated by increased collagen and extracellular matrix deposition and increased passive mechanical properties of muscle bundles. Overall, our data demonstrate that nesprin 1 and desmin serve redundant roles in nuclear anchorage and that the loss of nuclear anchorage in skeletal muscle results in a pathological response characterized by increased tissue fibrosis and mechanical stiffness. PMID:24943590

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

PubMed Central

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-01-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. PMID:23980694

Disruption of both nesprin 1 and desmin results in nuclear anchorage defects and fibrosis in skeletal muscle.

PubMed

Chapman, Mark A; Zhang, Jianlin; Banerjee, Indroneal; Guo, Ling T; Zhang, Zhiwei; Shelton, G Diane; Ouyang, Kunfu; Lieber, Richard L; Chen, Ju

2014-11-15

Proper localization and anchorage of nuclei within skeletal muscle is critical for cellular function. Alterations in nuclear anchoring proteins modify a number of cellular functions including mechanotransduction, nuclear localization, chromatin positioning/compaction and overall organ function. In skeletal muscle, nesprin 1 and desmin are thought to link the nucleus to the cytoskeletal network. Thus, we hypothesize that both of these factors play a key role in skeletal muscle function. To examine this question, we utilized global ablation murine models of nesprin 1, desmin or both nesprin 1 and desmin. Herein, we have created the nesprin-desmin double-knockout (DKO) mouse, eliminating a major fraction of nuclear-cytoskeletal connections and enabling understanding of the importance of nuclear anchorage in skeletal muscle. Globally, DKO mice are marked by decreased lifespan, body weight and muscle strength. With regard to skeletal muscle, DKO myonuclear anchorage was dramatically decreased compared with wild-type, nesprin 1(-/-) and desmin(-/-) mice. Additionally, nuclear-cytoskeletal strain transmission was decreased in DKO skeletal muscle. Finally, loss of nuclear anchorage in DKO mice coincided with a fibrotic response as indicated by increased collagen and extracellular matrix deposition and increased passive mechanical properties of muscle bundles. Overall, our data demonstrate that nesprin 1 and desmin serve redundant roles in nuclear anchorage and that the loss of nuclear anchorage in skeletal muscle results in a pathological response characterized by increased tissue fibrosis and mechanical stiffness. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fasudil, a clinically-safe ROCK Inhibitor, Decreases Disease Burden in a Cbl/Cbl-b Deficiency-Driven Murine Model of Myeloproliferative Disorders

PubMed Central

William, Basem M.; An, Wei; Feng, Dan; Nadeau, Scott; Mohapatra, Bhopal C; Storck, Matthew A.; Band, Vimla; Band, Hamid

2017-01-01

Objectives Mutations in Cbl or Cbl-b gene occur in 10% of MPD patients and are associated with poor prognosis. Hematopoietic Cbl/Cbl-b double knockout (DKO) leads to a disease in mice phenotypically similar to human MPDs. The aim of this study was to evaluate the anti-MPD activity of a clinical safe drug, Fasudil identified in an in vitro kinase inhibitor as an inhibitor of proliferation of DKO mouse hematopoietic stem/progenitor cells (HSPCs). Methods Fasudil exhibited relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow HSPCs. We established a mouse model with uniform time of MPD onset by transplanting Cbl/Cbl-b DKO HSPCs into busulfan-conditioned NOD/SCID/gamma chain-deficient mice. Four weeks post-transplant, mice were treated with 100 mg/kg fasudil (13 mice) or water (control, 8 mice) daily by oral gavage, followed by blood cell count every two weeks. Results By two weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil. We observed a trend towards improved survival in fasudil-treated mice that didn’t reach statistical significance. Notably, prolonged survival beyond 27 weeks was observed in 2 fasudil-treated mice, nearly twice the 16-week average life-span in the Cbl/Cbl-b DKO MPD model. Conclusions Our results suggest a therapeutic potential for fasudil, a clinically-safe drug with promising results in vascular diseases, in the treatment of MPDs or other mutant Cbl-driven myeloid disorders. PMID:26177294

Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion

PubMed Central

Martin, Gregory G.; Atshaves, Barbara P.; Landrock, Kerstin K.; Landrock, Danilo; Storey, Stephen M.; Howles, Philip N.; Kier, Ann B.

2014-01-01

On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol. PMID:25277800

TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE−/− mice

PubMed Central

Prasad, Sakamuri Siva Sankara Vara; Higashi, Yusuke; Sukhanov, Sergiy; Siddesha, Jalahalli M; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

2016-01-01

Background and aims Atherosclerosis is a major cause of heart attack and stroke. Inflammation plays a critical role in the development of atherosclerosis. Since the cytoplasmic adaptor molecule TRAF3IP2 (TRAF3-Interacting Protein 2) plays a causal role in various autoimmune and inflammatory diseases, we hypothesized that TRAF3IP2 mediates atherosclerotic plaque development. Methods TRAF3IP2/ApoE double knockout (DKO) mice were generated by crossing TRAF3IP2−/− and ApoE−/− mice. ApoE−/− mice served as controls. Both DKO and control mice were fed a high-fat diet for 12 weeks. Plasma lipids were measured by ELISA, atherosclerosis by en face analysis of aorta and plaque cross-section measurements at the aortic valve region, plaque necrotic core area, collagen and smooth muscle cell content by histomorphometry, and aortic gene expression by RT-qPCR. Results The plasma lipoprotein profile was not altered by TRAF3IP2 gene deletion in ApoE−/− mice. While total aortic plaque area was decreased in DKO female, but not male mice, the plaque necrotic area was significantly decreased in DKO mice of both genders. Plaque collagen and smooth muscle cell contents were increased significantly in both female and male DKO mice compared to respective controls. Aortic expression of proinflammatory cytokine (Tumor necrosis factor α, TNFα), chemokine (Chemokine (C-X-C motif) Ligand 1, CXCL1) and adhesion molecule (Vascular cell adhesion molecule 1, VCAM1; and Intercellular adhesion molecule 1, ICAM1) gene expression were decreased in both male and female DKO mice. In addition, the male DKO mice showed a markedly reduced expression of extracellular matrix (ECM)-related genes, including TIMP1 (Tissue inhibitor of metalloproteinase 1), RECK (Reversion-Inducing- Cysteine-Rich Protein with Kazal Motifs) and ADAM17 (A Disintegrin And Metalloproteinase 17). Conclusions TRAF3IP2 plays a causal role in atherosclerotic plaque development and vulnerability, possibly by inducing the expression of multiple proinflammatory mediators. TRAF3IP2 could be a potential therapeutic target in atherosclerotic vascular diseases. PMID:27237075

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

PubMed

Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

2016-10-01

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

PubMed

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-10-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

PubMed

Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

2017-01-01

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion.

PubMed

Martin, Gregory G; Atshaves, Barbara P; Landrock, Kerstin K; Landrock, Danilo; Storey, Stephen M; Howles, Philip N; Kier, Ann B; Schroeder, Friedhelm

2014-12-01

On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol. Copyright © 2014 the American Physiological Society.

A role for calmodulin-stimulated adenylyl cyclases in cocaine sensitization.

PubMed

DiRocco, Derek P; Scheiner, Zachary S; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R

2009-02-25

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1(-/-) and AC8(-/-)) exhibit Ca(2+)-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca(2+)-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs.

A Role for Calmodulin-Stimulated Adenylyl Cyclases in Cocaine Sensitization

PubMed Central

DiRocco, Derek P.; Scheiner, Zachary S.; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R.

2009-01-01

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that while AC1 and AC8 single knockout mice (AC1−/− and AC8−/−) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knockout (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization following chronic cocaine treatment. Because of the known role for the ERK/MAP kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated extracellular signal-regulated kinase (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase positive (ChAT+) interneurons in DKO mice relative to wild-type (WT) controls. Following acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581, and cAMP response element-binding protein (pCREB) at Ser133 following acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. PMID:19244515

Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration.

PubMed

Mayerl, Steffen; Schmidt, Manuel; Doycheva, Denica; Darras, Veerle M; Hüttner, Sören S; Boelen, Anita; Visser, Theo J; Kaether, Christoph; Heuer, Heike; von Maltzahn, Julia

2018-06-05

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Abrogated Freud-1/Cc2d1a Repression of 5-HT1A Autoreceptors Induces Fluoxetine-Resistant Anxiety/Depression-Like Behavior.

PubMed

Vahid-Ansari, Faranak; Daigle, Mireille; Manzini, M Chiara; Tanaka, Kenji F; Hen, René; Geddes, Sean D; Béïque, Jean-Claude; James, Jonathan; Merali, Zul; Albert, Paul R

2017-12-06

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo , we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons ( cF1ko ). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out ( cF1/1A dko ) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment. SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response. Copyright © 2017 the authors 0270-6474/17/3711967-12$15.00/0.

Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

PubMed

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

PubMed Central

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression. PMID:25486569

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

PubMed Central

MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

PubMed

Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Bile acid excess induces cardiomyopathy and metabolic dysfunctions in the heart

PubMed Central

Desai, Moreshwar; Mathur, Bhoomika; Eblimit, Zeena; Vasquez, Hernan; Taegtmeyer, Heinrich; Karpen, Saul; Penny, Daniel J.; Moore, David D.; Anakk, Sayeepriyadarshini

2017-01-01

Cardiac dysfunction in patients with liver cirrhosis is strongly associated with increased serum bile acid concentrations. Here we show that excess bile acids decrease fatty acid oxidation in cardiomyocytes and can cause heart dysfunction, a cardiac syndrome that we term Cholecardia. Fxr; Shp double knockout (DKO) mice, a model for bile acid overload, display cardiac hypertrophy, bradycardia, and exercise intolerance. In addition, DKO mice exhibit an impaired cardiac response to catecholamine challenge. Consistent with this decreased cardiac function, we show that elevated serum bile acids reduce cardiac fatty acid oxidation both in vivo and ex vivo. We find that increased bile acid levels suppress expression of Pgc1α, a key regulator of fatty acid metabolism, and that Pgc1α overexpression in cardiac cells was able to rescue the bile acid-mediated reduction in fatty acid oxidation genes. Importantly, intestinal bile acid sequestration with cholestyramine was sufficient to reverse the observed heart dysfunction in the DKO mice. Conclusions Overall, we propose that decreased Pgc1α expression contributes to the metabolic dysfunction in Cholecardia, and that reducing serum bile acid concentrations will be beneficial against metabolic and pathological changes in the heart. PMID:27774647

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE−/− mice

PubMed Central

Vujic, Nemanja; Korbelius, Melanie; Leopold, Christina; Duta-Mare, Madalina; Rainer, Silvia; Schlager, Stefanie; Goeritzer, Madeleine; Kolb, Dagmar; Eichmann, Thomas O.; Diwoky, Clemens; Zimmer, Andreas; Zimmermann, Robert; Lass, Achim; Radovic, Branislav; Kratky, Dagmar

2017-01-01

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (−/−) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)−/− mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system. PMID:28380440

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE-/- mice.

PubMed

Vujic, Nemanja; Korbelius, Melanie; Leopold, Christina; Duta-Mare, Madalina; Rainer, Silvia; Schlager, Stefanie; Goeritzer, Madeleine; Kolb, Dagmar; Eichmann, Thomas O; Diwoky, Clemens; Zimmer, Andreas; Zimmermann, Robert; Lass, Achim; Radovic, Branislav; Kratky, Dagmar

2017-05-16

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis.

PubMed

Shi, Hao; Gatzke, Florian; Molle, Julia M; Lee, Han Bin; Helm, Emma T; Oldham, Jessie J; Zhang, Lei; Gerrard, David E; Bennett, Anton M

2015-11-12

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis

PubMed Central

Shi, Hao; Gatzke, Florian; Molle, Julia M.; Lee, Han Bin; Helm, Emma T.; Oldham, Jessie J.; Zhang, Lei; Gerrard, David E.; Bennett, Anton M.

2015-01-01

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways. PMID:27064463

SLP-76 couples Syk to the osteoclast cytoskeleton.

PubMed

Reeve, Jennifer L; Zou, Wei; Liu, Yuli; Maltzman, Jonathan S; Ross, F Patrick; Teitelbaum, Steven L

2009-08-01

The capacity of the osteoclast (OC) to resorb bone is dictated by cytoskeletal organization, which in turn emanates from signals derived from the alpha(v)beta(3) integrin and c-Fms. Syk is key to these signals and, in other cells, this tyrosine kinase exerts its effects via intermediaries including the SLP adaptors, SLP-76 and BLNK (B cell linker). Thus, we asked whether these two SLP proteins regulate OC function. We find BLNK-deficient OCs are normal, whereas cytoskeletal organization of those lacking SLP-76 is delayed, thus modestly reducing bone resorption in vitro. Cytoskeletal organization and bone resorption are more profoundly arrested in cultured OCs deficient in BLNK and SLP-76 double knockout (DKO) phenotypes. In contrast, stimulated bone resorption in vivo is inhibited approximately 40% in either SLP-76(-/-) or DKO mice. This observation, taken with the fact that DKO OCs are rescued by retroviral transduction of only SLP-76, indicates that SLP-76 is the dominant SLP family member in the resorptive process. We also find SLP-76 is phosphorylated in a Syk-dependent manner. Furthermore, in the absence of the adaptor protein, integrin-mediated phosphorylation of Vav3, the OC cytoskeleton-organizing guanine nucleotide exchange factor, is abrogated. In keeping with a central role of SLP-76/Vav3 association in osteoclastic resorption, retroviral transduction of SLP-76, in which the Vav binding site is disrupted (3YF), fails to normalize the cytoskeleton of DKO OCs and the resorptive capacity of the cells. Finally, c-Fms-activated Syk also exerts its OC cytoskeleton-organizing effect in a SLP-76/Vav3-dependent manner.

Haematopoietic leptin receptor deficiency does not affect macrophage accumulation in adipose tissue or systemic insulin sensitivity.

PubMed

Gutierrez, Dario A; Hasty, Alyssa H

2012-03-01

The adipokine leptin is primarily produced by white adipose tissue (AT) and is a potent monocyte/macrophage chemoattractant in vitro. The long form of the leptin receptor (LepR) is required for monocyte/macrophage chemotaxis towards leptin. In this study, we examined the effects of haematopoietic LepR as well as LepR with C-C chemokine receptor 2 (CCR2) deficiency (double knockout (DKO)) on macrophage recruitment to AT after two different periods of high fat diet (HFD) feeding. Briefly, 8-week-old C57BL/6 mice were transplanted with bone marrow (BM) from Lepr(+/+), Lepr(-/-) or DKO donors (groups named BM-Lepr(+/+), BM-Lepr(-/-) and BM-DKO respectively), and were placed on an HFD for 6 or 12 weeks. At the end of the study, macrophage infiltration and the inflammatory state of AT were evaluated by real-time RT-PCR, histology and flow cytometry. In addition, glucose and insulin tolerance were assessed at both time points. Our results showed no differences in macrophage accumulation or AT inflammatory state between the BM-Lepr(+/+) and BM-Lepr(-/-) mice after 6 or 12 weeks of HFD feeding; any effects observed in the BM-DKO were attributed to the haematopoietic deficiency of CCR2. In addition, no changes in glucose or insulin tolerance were observed between groups after either period of HFD feeding. Our findings suggest that although leptin is a potent chemoattractant in vitro, haematopoietic LepR deficiency does not affect macrophage accumulation in AT in early to moderate stages of diet-induced obesity.

Toll-like receptors 2 and 4 exert opposite effects on the contractile response induced by serotonin in mouse colon: role of serotonin receptors.

PubMed

Forcén, R; Latorre, E; Pardo, J; Alcalde, A I; Murillo, M D; Grasa, L

2016-08-01

What is the central question of this study? The action of Toll-like receptors (TLRs) 2 and 4 on the motor response to serotonin in mouse colon has not previously been reported. What is the main finding and its importance? Toll-like receptors 2 and 4 modulate the serotonin-induced contractile response in mouse colon by modifying the expression of serotonin (5-HT) receptors. Alterations in 5-HT2A and 5-HT2C receptors explain the increase of the response to serotonin in TLR2(-/-) mice. Alterations in 5-HT2C and 5-HT4 receptors explain the suppression of the response to serotonin in TLR4(-/-) mice. The microbiota, through Toll-like receptors (TLRs), may regulate gastrointestinal motility by activating neuroendocrine mechanisms. We evaluated the influence of TLR2 and TLR4 in spontaneous contractions and in the serotonin (5-HT)-induced motor response in mouse colon, and assessed the 5-HT receptors involved. Muscle contractility studies to evaluate the intestinal spontaneous motility and the response to 5-HT were performed in the colon from wild-type (WT), TLR2(-/-) , TLR4(-/-) and TLR2/4 double knockout (DKO) mice. The 5-HT receptor mRNA expression was determined by real-time PCR. The amplitude and frequency of the spontaneous contractions of the colon were smaller in TLR4(-/-) and TLR2/4 DKO mice with respect to WT mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 100 μm 5-HT evoked a contractile response. The contractile response induced by 5-HT was significantly higher in TLR2(-/-) than in WT mice. In TLR4(-/-) mice, 5-HT did not evoke any contractile response. The mRNA expression of 5-HT2A was increased in TLR2(-/-) and TLR2/4 DKO mice. The 5-HT2C and 5-HT4 mRNA expressions were increased in TLR4(-/-) and TLR2/4 DKO mice. The 5-HT2C mRNA expression was diminished in TLR2(-/-) mice. The 5-HT3 mRNA expression was increased in TLR2(-/-) , TLR4(-/-) and TLR2/4 DKO mice. The 5-HT7 mRNA expression was diminished in TLR2/4 DKO mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 5-HT2 , 5-HT3 , 5-HT4 and 5-HT7 receptor antagonists reduced or blocked the contractile response evoked by 5-HT. We postulate that TLR2 and TLR4 modulate the serotonin contractile motor response in mouse colon in an opposing manner by modifying the expression of several serotonin receptors. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Fujimoto, Yuki; Furuie, Sumie; Yamamura, Ken-Ichi; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Moriyama, Mitsuaki; Sinasac, David S; Yamamoto, Takashi; Furukawa, Tatsuhiko; Kobayashi, Keiko

2017-04-01

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Synapsin- and Actin-Dependent Frequency Enhancement in Mouse Hippocampal Mossy Fiber Synapses

PubMed Central

Owe, Simen G.; Jensen, Vidar; Evergren, Emma; Ruiz, Arnaud; Shupliakov, Oleg; Kullmann, Dimitri M.; Storm-Mathisen, Jon; Walaas, S. Ivar; Hvalby, Øivind

2009-01-01

The synapsin proteins have different roles in excitatory and inhibitory synaptic terminals. We demonstrate a differential role between types of excitatory terminals. Structural and functional aspects of the hippocampal mossy fiber (MF) synapses were studied in wild-type (WT) mice and in synapsin double-knockout mice (DKO). A severe reduction in the number of synaptic vesicles situated more than 100 nm away from the presynaptic membrane active zone was found in the synapsin DKO animals. The ultrastructural level gave concomitant reduction in F-actin immunoreactivity observed at the periactive endocytic zone of the MF terminals. Frequency facilitation was normal in synapsin DKO mice at low firing rates (∼0.1 Hz) but was impaired at firing rates within the physiological range (∼2 Hz). Synapses made by associational/commissural fibers showed comparatively small frequency facilitation at the same frequencies. Synapsin-dependent facilitation in MF synapses of WT mice was attenuated by blocking F-actin polymerization with cytochalasin B in hippocampal slices. Synapsin III, selectively seen in MF synapses, is enriched specifically in the area adjacent to the synaptic cleft. This may underlie the ability of synapsin III to promote synaptic depression, contributing to the reduced frequency facilitation observed in the absence of synapsins I and II. PMID:18550596

Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy

PubMed Central

An, Wei; Nadeau, Scott A.; Mohapatra, Bhopal C.; Feng, Dan; Zutshi, Neha; Storck, Matthew D.; Arya, Priyanka; Talmadge, James E.; Meza, Jane L.; Band, Vimla; Band, Hamid

2015-01-01

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling. PMID:25871390

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

PubMed Central

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E.; Rajan, Sudarsan; Verma, Vipin K.; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R.; Muniswamy, Madesh; Kishore, Raj; Lal, Hind; Force, Thomas

2016-01-01

Rationale Cardiac myocyte-specific deletion of either Glycogen Synthase Kinase (GSK)3A or GSK3B leads to cardiac protection following myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration due to the fact that all GSK-3–targeted drugs including the drugs already in clinical trial target both isoforms of GSK-3 and none are isoform specific. Objective To identify the consequences of combined deletion of cardiac myocyte GSK3A and GSK3B in heart function. Methods and Results We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout, DKO). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, DKO hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from DKO implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. DKO cardiac myocytes showed cell cycle progression resulting in increased DNA content and multi-nucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Conclusion Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis and its loss is incompatible with life due to cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. PMID:26976650

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

PubMed Central

Cheng, Catherine; Nowak, Roberta B.; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K.; Lo, Woo-Kuen; Mathias, Richard T.

2015-01-01

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1−/−;CP49−/− double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. PMID:25740157

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

PubMed

Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K; Lo, Woo-Kuen; Mathias, Richard T; Fowler, Velia M

2015-05-15

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. Copyright © 2015 the American Physiological Society.

Engraftment of PBMC from SLE and APS Donors into BALB-Rag2−/−IL2Rgc−/− Mice: a Promising Model for Studying Human Disease

PubMed Central

Andrade, Danieli; Redecha, Patricia B.; Vukelic, Milena; Qing, Xiaoping; Perino, Giorgio; Salmon, Jane E.; Koo, Gloria C.

2011-01-01

Purpose To construct a humanized SLE mouse that resembles the human disease to define pathophysiology and targeted for treatments. Methods We infused peripheral blood mononuclear cells (PBMC) from SLE patients into BALB-Rag2−/−IL2Rgc−/−mice (DKO), which lack T, B and NK cells. PBMC from 5 SLE patients and 4 normal donors (ND) at 3–5×106/mouse were infused IV/IP to non-irradiated 4–5 weeks old mice. We evaluated the engraftment of human CD45+cells and monitored the plasma human IgG, anti-dsDNA, anti-cardiolipin (aCL) antibodies, proteinuria, and kidney histology. Results We found 100% successful engraftment of 40 DKO mice infused with human PBMC. In both SLE-DKO and ND-DKO mice, 50–80% human CD45+ cells were observed in PBMC fraction 4–6 weeks post engraftment, with 70–90% CD3+ cells. There were fewer CD3+4+cells (5.5±2.1%) and more CD3+8+cells (79.4±3.6%) in the SLE-DKO mice, as in the SLE patients. CD19+B cells and CD11c+Monocytic cells were found in the spleen, lung, liver and bone marrow. There was no significant difference in plasma human IgG levels and anti-dsDNA antibodies between SLE-DKO and ND-DKO mice. Levels of aCL antibody were significantly higher in all SLE-DKO mice infused with PBMC from a SLE patient with high titers of aCL antibodies. SLE-DKO mice had proteinuria, human IgG deposits in the kidneys and shorter life span. In SLE- DKO mice engrafted from the aCL-positive patient, we found micro-thrombi and infiltration of CD3+, CD8+ and CD19+ cells in the glomeruli, recapitulating APS in these mice. Conclusion A novel humanized SLE-DKO mouse is established, exhibiting many characteristics of immunologic and clinical features of SLE. PMID:21560114

Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

PubMed

Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

2014-01-01

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

PubMed

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-10-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

PubMed Central

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-01-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

PubMed Central

Ahmad, Gulzar; Mohapatra, Bhopal; Schulte, Nancy A.; Nadeau, Scott; Luan, Haitao; Zutshi, Neha; Tom, Eric; Ortega-Cava, Cesar; Tu, Chun; Sanada, Masashi; Ogawa, Seishi; Toews, Myron L.; Band, Vimla; Band, Hamid

2014-01-01

Members of the Casitas B-Lineage Lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK. PMID:25449262

Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice.

PubMed

Zhu, Xinmei; Ng, Hang Pong; Lai, Yen-Chun; Craigo, Jodi K; Nagilla, Pruthvi S; Raghani, Pooja; Nagarajan, Shanmugam

2014-09-01

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis. Copyright © 2014 by The American Association of Immunologists, Inc.

Immunoproteasome in animal models of Duchenne muscular dystrophy.

PubMed

Chen, Chiao-Nan Joyce; Graber, Ted G; Bratten, Wendy M; Ferrington, Deborah A; Thompson, LaDora V

2014-04-01

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

The transcriptional coactivators p/CIP and SRC-1 control insulin resistance through IRS1 in obesity models.

PubMed

Wang, Zhiyong; Shah, O Jameel; Hunter, Tony

2012-01-01

Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactivators for nuclear hormone receptors and other transcription factors in vitro. In a previous study, we reported initial characterization of the obesity-resistant phenotypes of p/CIP and SRC-1 double knockout (DKO) mice, which exhibit increased energy expenditure, and suggested that nuclear hormone receptor target genes were involved in these phenotypes. In this study, we demonstrate that p/CIP and SRC1 control insulin signaling in a cell-autonomous manner both in vitro and in vivo. Genetic deletion of p/CIP and SRC-1 increases glucose uptake and enhances insulin sensitivity in both regular chow- and high fat diet-fed DKO mice despite increased food intake. Interestingly, we discover that loss of p/CIP and SRC-1 results in resistance to age-related obesity and glucose intolerance. We show that expression levels of a key insulin signaling component, insulin receptor substrate 1 (IRS1), are significantly increased in two cell lines representing fat and muscle lineages with p/CIP and SRC-1 deletions and in white adipose tissue and skeletal muscle of DKO mice; this may account for increased glucose metabolism and insulin sensitivity. This is the first evidence that the p160 coactivators control insulin signaling and glucose metabolism through IRS1. Therefore, our studies indicate that p/CIP and SRC-1 are potential therapeutic targets not only for obesity but also for diabetes.

The Transcriptional Coactivators p/CIP and SRC-1 Control Insulin Resistance through IRS1 in Obesity Models

PubMed Central

Wang, Zhiyong; Shah, O. Jameel; Hunter, Tony

2012-01-01

Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactivators for nuclear hormone receptors and other transcription factors in vitro. In a previous study, we reported initial characterization of the obesity-resistant phenotypes of p/CIP and SRC-1 double knockout (DKO) mice, which exhibit increased energy expenditure, and suggested that nuclear hormone receptor target genes were involved in these phenotypes. In this study, we demonstrate that p/CIP and SRC1 control insulin signaling in a cell-autonomous manner both in vitro and in vivo. Genetic deletion of p/CIP and SRC-1 increases glucose uptake and enhances insulin sensitivity in both regular chow- and high fat diet-fed DKO mice despite increased food intake. Interestingly, we discover that loss of p/CIP and SRC-1 results in resistance to age-related obesity and glucose intolerance. We show that expression levels of a key insulin signaling component, insulin receptor substrate 1 (IRS1), are significantly increased in two cell lines representing fat and muscle lineages with p/CIP and SRC-1 deletions and in white adipose tissue and skeletal muscle of DKO mice; this may account for increased glucose metabolism and insulin sensitivity. This is the first evidence that the p160 coactivators control insulin signaling and glucose metabolism through IRS1. Therefore, our studies indicate that p/CIP and SRC-1 are potential therapeutic targets not only for obesity but also for diabetes. PMID:22859932

Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-05-16

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2014-01-01

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure.

PubMed

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-08-14

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

PubMed Central

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E.; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-01-01

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema. PMID:22847418

A saturation hypothesis to explain both enhanced and impaired learning with enhanced plasticity

PubMed Central

Nguyen-Vu, TD Barbara; Zhao, Grace Q; Lahiri, Subhaneil; Kimpo, Rhea R; Lee, Hanmi; Ganguli, Surya; Shatz, Carla J; Raymond, Jennifer L

2017-01-01

Across many studies, animals with enhanced synaptic plasticity exhibit either enhanced or impaired learning, raising a conceptual puzzle: how enhanced plasticity can yield opposite learning outcomes? Here, we show that the recent history of experience can determine whether mice with enhanced plasticity exhibit enhanced or impaired learning in response to the same training. Mice with enhanced cerebellar LTD, due to double knockout (DKO) of MHCI H2-Kb/H2-Db (KbDb−/−), exhibited oculomotor learning deficits. However, the same mice exhibited enhanced learning after appropriate pre-training. Theoretical analysis revealed that synapses with history-dependent learning rules could recapitulate the data, and suggested that saturation may be a key factor limiting the ability of enhanced plasticity to enhance learning. Optogenetic stimulation designed to saturate LTD produced the same impairment in WT as observed in DKO mice. Overall, our results suggest that the recent history of activity and the threshold for synaptic plasticity conspire to effect divergent learning outcomes. DOI: http://dx.doi.org/10.7554/eLife.20147.001 PMID:28234229

Comments on "Determination and Analysis of the Theoretical Production of a Bucket Wheel Excavator" / Uwagi I Komentarze Do Pracy: "Określanie I Analiza Teoretycznej Wydajności Pracy Koparki Wielonaczyniowej Kołowej"

NASA Astrophysics Data System (ADS)

Bošnjak, Srđan M.

2015-03-01

This paper comments on the recently published work dealing with the problem in the determination of the theoretical output of the bucket wheel excavator. It also includes remarks on the inadequacy in the problem approach and highlights the mistakes in the mathematical model. This work emphasizes the demand for a much wider and deeper approach to the problem of determining the output of the bucket wheel excavator. Opublikowany niedawno artykuł autorstwa Che i Chena (2014) poświecony jest ważnej kwestii jaką jest określenie teoretycznej wydajności koparki kołowej wielonaczyniowej. W załączonym przeglądzie literatury Che i Chen (2014) nie umieścili pozycji odnoszących się do metod urabiania, parametrów pracy koparki oraz teoretycznej wydajności wydobycia i być może to właśnie jest przyczyną pewnych niedokładności powstałych w trakcie rozwiązywania problemu. Intencją autora obecnej publikacji było: • Odniesienie się do krytycyzmu wyrażonego przez Che i Chena (2014) dotyczącego procedury obliczania prędkości w ruchu łukowym podanej w cytowanej literaturze przedmiotu (Pajer i in., 1971; Vetrov, 1971; Rasper, 1975; Durst i Vogt, 1988); • Zbadanie różnic pomiędzy procedurą określania teoretycznej wydajności zaproponowaną w pracy Che i Chena (2014) a odpowiednimi rozwiązaniami podanymi w literaturze; • Określenie prawidłowości i stosowalności teorii zaprezentowanej w pracy Che i Chena (2014) poprzez porównanie wyników uzyskanych z wykorzystaniem ich teorii oraz teorii podanych w wymienionych pozycjach literatury. W rozdziale 2 pracy (Che i Chen 2014) zatytułowanym " Uprzednio stosowane metody określania teoretycznej wydajności pracy koparki kołowej wielonaczyniowej" autorzy nie podali głównych odniesień literaturowych z których zaczerpnięte zostały równania (1)-(6)**. Ponadto, nie podali charakterystyki modelu działania koparki, na podstawie którego wyprowadzone zostały rzeczone równania, co stanowi poważne przeoczenie. Che i Chen (2014) kwestionują prawidłowość równania (6), lecz przedstawione w niniejszej pracy analizy i uwagi prowadzą do następujących wniosków: • Stwierdzenie w pracy (Che i Chen 2014) że równanie (6) nie uwzględnia wpływu tmax i h nie jest prawdziwe. Dowód: Równanie (6'). • Stwierdzenie że równanie (6) nie uwzględnia wpływu R_s^' - "...czyli obrotowego promienia działania..." (Che i Chen 2014) jest prawdziwe, w przeciwnym razie równanie (6) byłoby nieprawdziwe. Równanie (6) uwzględnia odpowiedni promień zdefiniowany w równaniu (i), które jest prawdziwe. Dowód: Równanie (6'). W rozdziale 2 pracy (Che i Chen 2014) zatytułowanym: "Analiza teoretycznej wydajności koparki kołowej wielonaczyniowej" kierując się błędnymi wnioskami odnośnie niedokładności równania (6) autorzy podjęli próbę skorygowania powyższej niedokładności, by w ten sposób poprawić istniejącą teorię, W trakcie realizacji koncepcji wykorzystania równania (11) do zdefiniowania zasad rządzących zmianą prędkości kątowej wysięgnika Che i Chen (2014) popełnili błąd. Przyjęli oni mianowicie, że wyrażenie (14), otrzymane dla górnego przekroju wykopu e odnosić się będzie także do wszystkich przekrojów, dla całej wysokości wrębu. Pominęli w ten sposób fakt iż prędkość w ruchu łukowym wysięgnika nie jest wielkością stałą, patrz: równania (f) i (f'), i przyjęli jej wartość maksymalną, równanie (f'). Dlatego też równania (15), (16), (18) i (19) nie są prawidłowe. Wszystkie wejściowe dane obliczeniowe a także sposób ich zapisu (Tabela 1) zostały zaczerpnięte z pracy (Che i Chen 2014). Ponadto, zaprezentowane dane o pracy koparki wielonaczyniowej SchRs wykorzystywanej w kopalni "Zukunft" w Reńskim Zagłębiu Węglowym, zaczerpnięto z pracy (Durst i Vogt 1988). Osiągnięcia badań teoretycznych Che i Chen (2014, strona 289) przedstawione dla konkretnej koparki wielonaczyniowej kołowej eksploatowanej w kopalni odkrywkowej lignitu w Reńskim Zagłębiu Węglowym ..." (Che i Chen 2014). Obliczenia głębokości wrębu i prędkości kątowej wysięgnika oraz teoretycznej wydajności pracy przeprowadzono na cztery sposoby, co obrazuje Tabela 2: • Wariant 1 - z wykorzystaniem teorii rozwiniętej przez Che i Chena (2014, strona 289); • Wariant 2 różni się od Wariantu 1 tylko zasadą określającą zmiany prędkości kątowej, i.e. określona jest ona wzorem: "...zasada 1/cosφ (Che i Chen 2014, strona 289); • Wariant 3 wykorzystuje znane równania podane w cytowanej literaturze przedmiotu (Durst i Vogt, 1988; Pajer i in., 1971; Rasper, 1975); • Wariant 4 różni od Wariantu 3 wyłącznie wyrażenie określające głębokości wrębu, w Wariancie 4 zastosowane zostało wyrażenie pozwalające na dokładne obliczenie głębokości wrębu. Zaprezentowane wyniki prowadzą do następujących wniosków: • Maksymalna teoretycznie obliczona wydajność dla wszystkich wariantów jest niższa od wartości maksymalnej otrzymanej przy wykorzystaniu równania (s), tak więc nie zachodzi niebezpieczeństwo nadmiernego przeciążenia łyżki, jak to zostało stwierdzone w pracy Che i Chena (2014); • Maksymalne teoretycznie wyliczone wydajności dla wariantu 1 i 2 należy odrzucić z powodu błędu w definicji promienia odniesienia Re, określonego równaniem (I), co zostało szczegółowo omówione w rozdziale 3 tej pracy; • Gdy teoretyczna wydajność obliczana jest w oparciu o dokładny wzór na głębokość wrębu (Wariant 4), otrzymujemy nieco wyższe wartości niż w Wariancie 3. Procentowo, różnica ta jest mniejsza niż 1% dla 0 ≤ φ ≤ 50.8°, poniżej 2% dla 0 ≤ φ ≤ 64.5° i poniżej 3% dla 0 ≤ φ ≤ 75.6° (rys. 11). Maksymalna wartość (5.9%) osiągana jest dla φ = 80°. Z punktu widzenia zastosowań inżynierskich, taki poziom błędu jest dopuszczalny, zwłaszcza gdy uwzględni się jego rozkład w zakresie 0 ≤ φ ≤ 80°, oraz fakt że błąd ten sprawia, że ryzyko zagrożenia maleje. • Obniżenie teoretycznej wydajności po tym jak prędkość kątowa osiąga wartość maksymalną (rys. 9): ".... jest akceptowalne ponieważ odnosi się do niewielkiej tylko części całkowitej objętości przenoszonego materiału" (Durst & Vogt, 1988, strona 46). Publikacja autorstwa Che i Chena (2014) jest godną uwagi próbą ulepszenia istniejącej teorii opisującej działanie koparek kołowych wielonaczyniowych. Jednakże, uwzględniając przedstawione uwagi i komentarze, nasuwają się następujące spostrzeżenia: • Badany problem jest nie jest problemem nowym i bogata literatura przedmiotu powinna była zostać uwzględniona; • Matematyczne wzory zawierają błędy, które nieuchronnie prowadzą do nieprawidłowych wniosków. Celem niniejszej pracy było zwrócenie uwagi na potrzebę zastosowania znacznie szerszego podejścia do problemu obliczania wydajności koparek kołowych wielonaczyniowych niż podejście zaproponowane w pracy Che i Chena (2014).

CRTC1 Function During Memory Encoding Is Disrupted in Neurodegeneration.

PubMed

Parra-Damas, Arnaldo; Chen, Meng; Enriquez-Barreto, Lilian; Ortega, Laura; Acosta, Sara; Perna, Judith Camats; Fullana, M Neus; Aguilera, José; Rodríguez-Alvarez, José; Saura, Carlos A

2017-01-15

Associative memory impairment is an early clinical feature of dementia patients, but the molecular and cellular mechanisms underlying these deficits are largely unknown. In this study, we investigated the functional regulation of the cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) by associative learning in physiological and neurodegenerative conditions. We evaluated the activation of CRTC1 in the hippocampus of control mice and mice lacking the Alzheimer's disease-linked presenilin genes (presenilin conditional double knockout [PS cDKO]) after one-trial contextual fear conditioning by using biochemical, immunohistochemical, and gene expression analyses. PS cDKO mice display classical features of neurodegeneration occurring in Alzheimer's disease including age-dependent cortical atrophy, neuron loss, dendritic degeneration, and memory deficits. Context-associative learning, but not single context or unconditioned stimuli, induces rapid dephosphorylation (Ser151) and translocation of CRTC1 from the cytosol/dendrites to the nucleus of hippocampal neurons in the mouse brain. Accordingly, context-associative learning induces differential CRTC1-dependent transcription of c-fos and the nuclear receptor subfamily 4 (Nr4a) genes Nr4a1-3 in the hippocampus through a mechanism that involves CRTC1 recruitment to CRE promoters. Deregulation of CRTC1 dephosphorylation, nuclear translocation, and transcriptional function are associated with long-term contextual memory deficits in PS cDKO mice. Importantly, CRTC1 gene therapy in the hippocampus ameliorates context memory and transcriptional deficits and dendritic degeneration despite ongoing cortical degeneration in this neurodegeneration mouse model. These findings reveal a critical role of CRTC1 in the hippocampus during associative memory, and provide evidence that CRTC1 deregulation underlies memory deficits during neurodegeneration. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Regulatory Role of the NF-kB Pathway in Lymphangiogenesis and Breast Cancer Metastasis

DTIC Science & Technology

2010-07-01

with anti - LYVE-1 and anti -VEGFR-3 or anti -Prox1 antibodies in serial sections of p50 KO and WT lungs, showing reduced lymphatic vessel density...3 protein as determined by MFI analysis of slides double-stained with anti -VEGFR-3 and anti -LYVE-1 antibodies (Figure 2). These data indicate that...expression of VEGFR-3 and LYVE-1 on liver endothelial cells compared with WT. (A) Livers of p50 KO and WT mice were double immunostained with anti -VEGFR

Absence of SOCS3 in the cardiomyocyte increases mortality in a gp130 dependent manner accompanied by contractile dysfunction and ventricular arrhythmias

PubMed Central

Yajima, Toshitaka; Murofushi, Yoshiteru; Zhou, Hanbing; Park, Stanley; Housman, Jonathan; Zhong, Zhao-Hua; Nakamura, Michinari; Machida, Mitsuyo; Hwang, Kyung-Kuk; Gu, Yusu; Dalton, Nancy D.; Yajima, Tomoko; Yasukawa, Hideo; Peterson, Kirk L; Knowlton, Kirk U.

2011-01-01

Background Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. Methods and Results We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT and p38) from 15 weeks of age and developed cardiac dysfunction from around 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca2+ transients were significantly increased in SOCS3 cKO failing hearts compared to wild-type (WT) hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca2+ sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO non-failing hearts accompanied by a sarcoplasmic reticulum Ca2+ overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double knockout mice. Double KO mice lived significantly longer and had different histological abnormalities when compared to SOCS3 cKO mice; thus, demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. Conclusions Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias. PMID:22082679

Current Translational Research and Murine Models For Duchenne Muscular Dystrophy

PubMed Central

Rodrigues, Merryl; Echigoya, Yusuke; Fukada, So-ichiro; Yokota, Toshifumi

2016-01-01

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscle degeneration. Mutations in the DMD gene result in the absence of dystrophin, a protein required for muscle strength and stability. Currently, there is no cure for DMD. Since murine models are relatively easy to genetically manipulate, cost effective, and easily reproducible due to their short generation time, they have helped to elucidate the pathobiology of dystrophin deficiency and to assess therapies for treating DMD. Recently, several murine models have been developed by our group and others to be more representative of the human DMD mutation types and phenotypes. For instance, mdx mice on a DBA/2 genetic background, developed by Fukada et al., have lower regenerative capacity and exhibit very severe phenotype. Cmah-deficient mdx mice display an accelerated disease onset and severe cardiac phenotype due to differences in glycosylation between humans and mice. Other novel murine models include mdx52, which harbors a deletion mutation in exon 52, a hot spot region in humans, and dystrophin/utrophin double-deficient (dko), which displays a severe dystrophic phenotype due the absence of utrophin, a dystrophin homolog. This paper reviews the pathological manifestations and recent therapeutic developments in murine models of DMD such as standard mdx (C57BL/10), mdx on C57BL/6 background (C57BL/6-mdx), mdx52, dystrophin/utrophin double-deficient (dko), mdxβgeo, Dmd-null, humanized DMD (hDMD), mdx on DBA/2 background (DBA/2-mdx), Cmah-mdx, and mdx/mTRKO murine models. PMID:27854202

Parp1 activation in mouse embryonic fibroblasts promotes Pol β-dependent cellular hypersensitivity to alkylation damage

PubMed Central

Jelezcova, Elena; Trivedi, Ram N.; Wang, Xiao-hong; Tang, Jiang-bo; Brown, Ashley R.; Goellner, Eva M.; Schamus, Sandy; Fornsaglio, Jamie L.; Sobol, Robert W.

2010-01-01

Alkylating agents induce cell death in wild-type (WT) mouse embryonic fibroblasts (MEFs) by multiple mechanisms, including apoptosis, autophagy and necrosis. DNA polymerase β (Pol β) knockout (KO) MEFs are hypersensitive to the cytotoxic effect of alkylating agents, as compared to WT MEFs. To test the hypothesis that Parp1 is preferentially activated by methyl methanesulfonate (MMS) exposure of Pol β KO MEFs, we have examined the relationship between Pol β expression, Parp1 activation and cell survival following MMS exposure in a series of WT and Pol β deficient MEF cell lines. Consistent with our hypothesis, we observed elevated Parp1 activation in Pol β KO MEFs as compared to matched WT MEFs. Both the MMS-induced activation of Parp1 and the MMS-induced cytoxicity of Pol β KO MEFs are attenuated by pre-treatment with the Parp1/Parp2 inhibitor PJ34. Further, elevated Parp1 activation is observed following knockdown (KD) of endogenous Pol β, as compared to WT cells. Pol β KD MEFs are hypersensitive to MMS and both the MMS-induced hypersensitivity and Parp1 activation is prevented by pre-treatment with PJ34. In addition, the MMS-induced cellular sensitivity of Pol β KO MEFs is reversed when Parp1 is also deleted (Pol β/Parp1 double KO MEFs) and we observe no MMS sensitivity differential between Pol β/Parp1 double KO MEFs and those that express recombinant mouse Pol β. These studies suggest that Parp1 may function as a sensor of BER to initiate cell death when BER is aborted or fails. Parp1 may therefore function in BER as a tumor suppressor by initiating cell death and preventing the accumulation of cells with chromosomal damage due to a BER defect. PMID:20096707

Chronic liver inflammation and hepatocellular carcinogenesis are independent of S100A9.

PubMed

De Ponti, Aurora; Wiechert, Lars; Stojanovic, Ana; Longerich, Thomas; Marhenke, Silke; Hogg, Nancy; Vogel, Arndt; Cerwenka, Adelheid; Schirmacher, Peter; Hess, Jochen; Angel, Peter

2015-05-15

The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC). Apart from this evidence, the role of calprotectin in hepatocyte transformation and tumor microenvironment is still unknown. The aim of this study was to define the function of S100A8/A9 in inflammation-driven HCC. Mice lacking S100a9 were crossed with the Mdr2(-/-) model, a prototype of inflammation-induced HCC formation. S100a9(-/-) Mdr2(-/-) (dKO) mice displayed no significant differences in tumor incidence or multiplicity compared to Mdr2(-/-) animals. Chronic liver inflammation, fibrosis and oval cell activation were not affected upon S100a9 deletion. Our data demonstrate that, although highly upregulated, calprotectin is dispensable in the onset and development of HCC, and in the maintenance of liver inflammation. © 2014 UICC.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

PubMed

Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

2016-06-01

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Repair of exogenous DNA double-strand breaks promotes chromosome synapsis in SPO11-mutant mouse meiocytes, and is altered in the absence of HORMAD1.

PubMed

Carofiglio, Fabrizia; Sleddens-Linkels, Esther; Wassenaar, Evelyne; Inagaki, Akiko; van Cappellen, Wiggert A; Grootegoed, J Anton; Toth, Attila; Baarends, Willy M

2018-03-01

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11 YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11 YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11 YF/YF compared to Spo11 +/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11 YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Skeletal muscle fibrosis in the mdx/utrn+/- mouse validates its suitability as a murine model of Duchenne muscular dystrophy.

PubMed

Gutpell, Kelly M; Hrinivich, William T; Hoffman, Lisa M

2015-01-01

Various therapeutic approaches have been studied for the treatment of Duchenne muscular dystrophy (DMD), but none of these approaches have led to significant long-term effects in patients. One reason for this observed inefficacy may be the use of inappropriate animal models for the testing of therapeutic agents. The mdx mouse is the most widely used murine model of DMD, yet it does not model the fibrotic progression observed in patients. Other murine models of DMD are available that lack one or both alleles of utrophin, a functional analog of dystrophin. The aim of this study was to compare fibrosis and myofiber damage in the mdx, mdx/utrn+/- and double knockout (dko) mouse models. We used Masson's trichrome stain and percentage of centrally-nucleated myofibers as indicators of fibrosis and myofiber regeneration, respectively, to assess disease progression in diaphragm and gastrocnemius muscles harvested from young and aged wild-type, mdx, mdx/utrn+/- and dko mice. Our results indicated that eight week-old gastrocnemius muscles of both mdx/utrn+/- and dko hind limb developed fibrosis whereas age-matched mdx gastrocnemius muscle did not (p = 0.002). The amount of collagen found in the mdx/utrn+/- diaphragm was significantly higher than that found in the corresponding diaphragm muscles of wild-type animals, but not of mdx animals (p = 0.0003). Aged mdx/utrn+/- mice developed fibrosis in both diaphragm and gastrocnemius muscles compared to wild-type controls (p = 0.003). Mdx diaphragm was fibrotic in aged mice as well (p = 0.0235), whereas the gastrocnemius muscle in these animals was not fibrotic. We did not measure a significant difference in collagen staining between wild-type and mdx gastrocnemius muscles. The results of this study support previous reports that the moderately-affected mdx/utrn+/- mouse is a better model of DMD, and we show here that this difference is apparent by 2 months of age.

The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6−/− shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand

PubMed Central

Conte, Daniele; Garaffo, Giulia; Lo Iacono, Nadia; Mantero, Stefano; Piccolo, Stefano; Cordenonsi, Michelangelo; Perez-Morga, David; Orecchia, Valeria; Poli, Valeria; Merlo, Giorgio R.

2016-01-01

The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5–DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered β-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology. PMID:26685160

Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1

PubMed Central

Huang, Yuning; Mizel, Diane

2013-01-01

Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I125-iothalamate (io) clearance. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/Piothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2−/−, AQP1−/−, and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; P < 0.01 vs. WT), 36.8 ± 1 (63 tubules; P < 0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; P < 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2−/− mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control. PMID:24049145

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

PubMed

Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S; McMurray, John S; Gandhi, Varsha

2016-01-19

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice.

PubMed

Zhang, Hong-Mei; Zhou, Hong-Yi; Chen, Shao-Rui; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2007-12-01

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

Bone Is a Major Target of PTH/PTHrP Receptor Signaling in Regulation of Fetal Blood Calcium Homeostasis

PubMed Central

Hirai, Takao; Kobayashi, Tatsuya; Nishimori, Shigeki; Karaplis, Andrew C.; Goltzman, David

2015-01-01

The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level. PMID:26052897

LOSS OF L-FABP, SCP-2/SCP-X, OR BOTH INDUCES HEPATIC LIPID ACCUMULATION IN FEMALE MICE

PubMed Central

Martin, Gregory G.; Atshaves, Barbara P.; Landrock, Kerstin K.; Landrock, Danilo; Schroeder, Friedhelm; Kier, Ann B.

2015-01-01

Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals—suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO- and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD). PMID:26116377

Cold Temperature Encoding by Cutaneous TRPA1 and TRPM8-Carrying Fibers in the Mouse

PubMed Central

Winter, Zoltan; Gruschwitz, Philipp; Eger, Stephanie; Touska, Filip; Zimmermann, Katharina

2017-01-01

Previous research identified TRPM8 and TRPA1 cold transducers with separate functions, one being functional in the non-noxious range and the second one being a nociceptive transducer. TRPM8-deficient mice present overt deficits in the detection of environmental cool, but not a lack of cold avoidance and TRPA1-deficient mice show clear deficits in some cold nocifensive assays. The extent of TRPA1's contribution to cold sensing in vivo is still unclear, because mice lacking both TRPM8 and TRPA1 (DKO) were described with unchanged cold avoidance from TRPM8−/− based on a two-temperature-choice assay and by c-fos measurement. The present study was designed to differentiate how much TRPM8 alone and combined TRPA1 and TRPM8 contribute to cold sensing. We analyzed behavior in the thermal ring track assay adjusted between 30 and 5°C and found a large reduction in cold avoidance of the double knockout mice as compared to the TRPM8-deficient mice. We also revisited skin-nerve recordings from saphenous-nerve skin preparations with regard to nociceptors and thermoreceptors. We compared the frequency and characteristics of the cold responses of TRPM8-expressing and TRPM8-negative C-fiber nociceptors in C57BL/6J mice with nociceptors of TRPM8-deficient and DKO mice and found that TRPM8 enables nociceptors to encode cold temperatures with higher firing rates and larger responses with sustained, static component. In TRPM8−/−, C-fiber cold nociceptors were markedly reduced and appeared further reduced in DKO. Nevertheless, the remaining cold responses in both knockout strains were similar in their characteristics and they were indifferent from the TRPM8-negative cold responses found in C57BL/6J mice. TRPM8 had a comparably essential role for encoding cold in thermoreceptors and lack of TRPM8 reduced response magnitude, peak and mean firing rates and the incidence of thermoreceptors. The encoding deficits were similar in the DKO strain. Our data illustrate that lack of TRPA1 in TRPM8-deficient mice results in a disproportionately large reduction in cold avoidance behavior and also affects the incidence of cold encoding fiber types. Presumably TRPA1 compensates for lack of TRPM8 to a certain extent and both channels cooperate to cover the entire cold temperature range, making cold-temperature encoding by TRPA1—although less powerful—synergistic to TRPM8. PMID:28713241

High-resolution vascular tissue characterization in mice using 55 MHz ultrasound hybrid imaging

PubMed Central

Mahmoud, Ahmed M.; Sandoval, Cesar; Teng, Bunyen; Schnermann, Jurgen B.; Martin, Karen H.; Mustafa, S. Jamal; Mukdadi, Osama M.

2012-01-01

Ultrasound and Duplex ultrasonography in particular are routinely used to diagnose cardiovascular disease (CVD), which is the leading cause of morbidity and mortality worldwide. However, these techniques may not be able to characterize vascular tissue compositional changes due to CVD. This work describes an ultrasound-based hybrid imaging technique that can be used for vascular tissue characterization and the diagnosis of atherosclerosis. Ultrasound radiofrequency (RF) data were acquired and processed in time, frequency, and wavelet domains to extract six parameters including time integrated backscatter (TIB), time variance (Tvar), time entropy (TE), frequency integrated backscatter (FIB), wavelet root mean square value (Wrms), and wavelet integrated backscatter (WIB). Each parameter was used to reconstruct an image co-registered to morphological B-scan. The combined set of hybrid images were used to characterize vascular tissue in vitro and in vivo using three mouse models including control (C57BL/6), and atherosclerotic apolipoprotein E-knockout (APOE-KO) and APOE/A1 adenosine receptor double knockout (DKO) mice. The technique was tested using high-frequency ultrasound including single-element (center frequency = 55 MHz) and commercial array (center frequency = 40 MHz) systems providing superior spatial resolutions of 24 μm and 40 μm, respectively. Atherosclerotic vascular lesions in the APOE-KO mouse exhibited the highest values (contrast) of −10.11 ± 1.92 dB, −12.13 ± 2.13 dB, −7.54 ± 1.45 dB, −5.10 ± 1.06 dB, −5.25 ± 0.94 dB, and −10.23 ± 2.12 dB in TIB, Tvar, TE, FIB, Wrms, WIB hybrid images (n = 10, p < 0.05), respectively. Control segments of normal vascular tissue showed the lowest values of −20.20 ± 2.71 dB, −22.54 ± 4.54 dB, −14.94 ± 2.05 dB, −9.64 ± 1.34 dB, −10.20 ± 1.27 dB, and −19.36 ± 3.24 dB in same hybrid images (n = 6, p < 0.05). Results from both histology and optical images showed good agreement with ultrasound findings within a maximum error of 3.6% in lesion estimation. This study demonstrated the feasibility of a high-resolution hybrid imaging technique to diagnose atherosclerosis and characterize plaque components in mouse. In the future, it can be easily implemented on commercial ultrasound systems and eventually translated into clinics as a screening tool for atherosclerosis and the assessment of vulnerable plaques. PMID:23218908

The "Don't Know" Option in Progress Testing

ERIC Educational Resources Information Center

Ravesloot, C. J.; Van der Schaaf, M. F.; Muijtjens, A. M. M.; Haaring, C.; Kruitwagen, C. L. J. J.; Beek, F. J. A.; Bakker, J.; Van Schaik, J.P.J.; Ten Cate, Th. J.

2015-01-01

Formula scoring (FS) is the use of a don't know option (DKO) with subtraction of points for wrong answers. Its effect on construct validity and reliability of progress test scores, is subject of discussion. Choosing a DKO may not only be affected by knowledge level, but also by risk taking tendency, and may thus introduce construct-irrelevant…

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

PubMed

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.

CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury

PubMed Central

Weinreuter, Martin; Kreusser, Michael M; Beckendorf, Jan; Schreiter, Friederike C; Leuschner, Florian; Lehmann, Lorenz H; Hofmann, Kai P; Rostosky, Julia S; Diemert, Nathalie; Xu, Chang; Volz, Hans Christian; Jungmann, Andreas; Nickel, Alexander; Sticht, Carsten; Gretz, Norbert; Maack, Christoph; Schneider, Michael D; Gröne, Hermann-Josef; Müller, Oliver J; Katus, Hugo A; Backs, Johannes

2014-01-01

CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKIIδC was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed, nor in cardiomyocyte-specific CaMKIIδ/γ double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1α, MIP-1α). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes. PMID:25193973

Thyroid function in mice with compound heterozygous and homozygous disruptions of SRC-1 and TIF-2 coactivators: evidence for haploinsufficiency.

PubMed

Weiss, Roy E; Gehin, Martine; Xu, Jianming; Sadow, Peter M; O'Malley, Bert W; Chambon, Pierre; Refetoff, Samuel

2002-04-01

Steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)-2 are homologous nuclear receptor coactivators. We have investigated their possible redundancy as thyroid hormone (TH) coactivators by measuring thyroid function in compound SRC-1 and TIF-2 knock out (KO) mice. Whereas SRC-1 KO (SRC-1(-/-)) mice are resistant to TH and SRC-1(+/-) are not, we now demonstrate that TIF-2 KO (TIF-2(-/-)) mice have normal thyroid function. Yet double heterozygous, SRC-1(+/-)/TIF-2(+/-) mice manifested resistance to TH of a similar degree as that in mice completely deficient in SRC-1. KO of both SRC-1 and TIF-2 resulted in marked increases of serum TH and thyrotropin concentrations. This work demonstrates gene dosage effect in nuclear coactivators manifesting as haploinsufficiency and functional redundancy of SRC-1 and TIF-2.

The Pure Rotational Spectrum of KO

NASA Astrophysics Data System (ADS)

Burton, Mark; Russ, Benjamin; Sheridan, Phillip M.; Bucchino, Matthew; Ziurys, Lucy M.

2017-06-01

The pure rotational spectrum of potassium monoxide (KO) has been recorded using millimeter-wave direct absorption spectroscopy. KO was synthesized by the reaction of potassium vapor, produced in a Broida-type oven, with nitrous oxide. No DC discharge was necessary. Eleven rotational transitions belonging to the ^{2}Π_{3/2} spin-orbit component have been measured and have been fit successfully to a case (c) Hamiltonian. Rotational and lambda-doubling constants for this spin-orbit component have been determined. It has been suggested that the ground electronic state of KO is either ^{2}Π (as for LiO and NaO) or ^{2}Σ (as for RbO and CsO), both of which lie close in energy. Recent computational studies favor a ^{2}Σ ground state. Further measurements of the rotational transitions of the ^{2}Π_{1/2} spin-orbit component and the ^{2}Σ state are currently in progress, as well as the potassium hyperfine structure.

Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-12-24

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord. PMID:20940295

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

PubMed

Schön, Christian; Asteriti, Sabrina; Koch, Susanne; Sothilingam, Vithiyanjali; Garcia Garrido, Marina; Tanimoto, Naoyuki; Herms, Jochen; Seeliger, Mathias W; Cangiano, Lorenzo; Biel, Martin; Michalakis, Stylianos

2016-03-15

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization

PubMed Central

Kopp, Hans-Georg; Hooper, Andrea T.; Broekman, M. Johan; Avecilla, Scott T.; Petit, Isabelle; Luo, Min; Milde, Till; Ramos, Carlos A.; Zhang, Fan; Kopp, Tabitha; Bornstein, Paul; Jin, David K.; Marcus, Aaron J.; Rafii, Shahin

2006-01-01

Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell–derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis. PMID:17143334

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

PubMed

Sano, Akiko; Matsushita, Hiroaki; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

2013-01-01

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

IL-23 is critical in the induction but not in the effector phase of experimental autoimmune encephalomyelitis.

PubMed

Thakker, Paresh; Leach, Michael W; Kuang, Wen; Benoit, Stephen E; Leonard, John P; Marusic, Suzana

2007-02-15

Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CNS, is a rodent model of human multiple sclerosis. IL-23 is one of the critical cytokines in EAE development and is currently believed to be involved in the maintenance of encephalitogenic responses during the tissue damage effector phase of the disease. In this study, we show that encephalitogenic T cells from myelin oligodendrocyte glycopeptide (MOG)-immunized wild-type (WT) mice caused indistinguishable disease when adoptively transferred to WT or IL-23-deficient (p19 knockout (KO)) recipient mice, demonstrating that once encephalitogenic cells have been generated, EAE can develop in the complete absence of IL-23. Furthermore, IL-12/23 double-deficient (p35/p19 double KO) recipient mice developed EAE that was indistinguishable from WT recipients, indicating that IL-12 did not compensate for IL-23 deficiency during the effector phase of EAE. In contrast, MOG-specific T cells from p19KO mice induced EAE with delayed onset and much lower severity when transferred to WT recipient mice as compared with the EAE that was induced by cells from WT controls. MOG-specific T cells from p19KO mice were highly deficient in the production of IFN-gamma, IL-17A, and TNF, indicating that IL-23 plays a critical role in development of encephalitogenic T cells and facilitates the development of T cells toward both Th1 and Th17 pathways.

NPY controls fear conditioning and fear extinction by combined action on Y₁ and Y₂ receptors.

PubMed

Verma, D; Tasan, R O; Herzog, H; Sperk, G

2012-06-01

Neuropeptide Y (NPY) and its receptors have been implicated in the control of emotional-affective processing, but the mechanism is unclear. While it is increasingly evident that stimulation of Y₁ and inhibition of Y₂ receptors produce prominent anxiolytic and antidepressant effects, the contribution of the individual NPY receptor subtypes in the acquisition and extinction of learned fear are unknown. Here we performed Pavlovian fear conditioning and extinction in NPY knockout (KO) and in NPY receptor KO mice. NPY KO mice display a dramatically accelerated acquisition of conditioned fear. Deletion of Y₁ receptors revealed only a moderately accelerated acquisition of conditioned fear, while lack of Y₂ receptors was without any effect on fear learning. However, the strong phenotype seen in NPY KO mice was reproduced in mice lacking both Y₁ and Y₂ receptors. In addition, NPY KO mice showed excessive recall of conditioned fear and impaired fear extinction. This behaviour was replicated only after deletion of both Y₁ and Y₂ receptors. In Y₁ receptor single KO mice, fear extinction was delayed and was unchanged in Y₂ receptor KO mice. Deletion of NPY and particularly Y₂ receptors resulted in a generalization of conditioned fear. Our data demonstrate that NPY delays the acquisition, reduces the expression of conditioned fear while promoting fear extinction. Although these effects appear to be primarily mediated by Y₁ receptors, the pronounced phenotype of Y₁Y₂ receptor double KO mice suggests a synergistic role of Y₂ receptors in fear acquisition and in fear extinction. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

2 p -insulator heterointerfaces: Creation of half-metallicity and anionogenic ferromagnetism via double exchange

NASA Astrophysics Data System (ADS)

Zhang, Baomin; Cao, Chonglong; Li, Guowei; Li, Feng; Ji, Weixiao; Zhang, Shufeng; Ren, Miaojuan; Zhang, Haikun; Zhang, Rui-Qin; Zhong, Zhicheng; Yuan, Zhe; Yuan, Shengjun; Blake, Graeme R.

2018-04-01

We use first-principles calculations to predict the occurrence of half-metallicity and anionogenic ferromagnetism at the heterointerface between two 2p insulators, taking the KO2/BaO2 (001) interface as an example. Whereas a sharp heterointerface is semiconducting, a heterointerface with a moderate concentration of swapped K and Ba atoms is half-metallic and ferromagnetic at ambient pressure due to the double exchange mechanism. The K-Ba swap renders the interfacial K-O and Ba-O atomic layers electron-doped and hole-doped, respectively. Our findings pave the way to realize metallicity and ferromagnetism at the interface between two 2 p insulators, and such systems can constitute a new family of heterostructures with novel properties, expanding studies on heterointerfaces from 3 d insulators to 2 p insulators.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

PubMed

Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

2016-01-01

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future. © 2015 Wiley Periodicals, Inc.

Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli

PubMed Central

Hu, Fengqi; Ding, Guohua; Zhang, Zhiyong; Gatto, Louis A.; Hawgood, Samuel; Poulain, Francis R.; Cooney, Robert N.; Wang, Guirong

2015-01-01

To investigate the effects of surfactant proteins A and D (SP-A, SP-D) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared to control, level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than infected WT mice 24 and 48 h post-infection. Basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher compared to WT mice. Phosphorylated-p38 level was elevated in the kidney of WT mice post-infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI. PMID:26511057

Development of mice without Cip/Kip CDK inhibitors.

PubMed

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu; Hara, Eiji; Nakayama, Keiko; Nakayama, Keiichi I

2012-10-19

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage). Copyright © 2012 Elsevier Inc. All rights reserved.

A critical role of fatty acid binding protein 4 and 5 (FABP4/5) in the systemic response to fasting.

PubMed

Syamsunarno, Mas Rizky A A; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; Tsushima, Yoshito; Endo, Keigo; Hotamisligil, Gökhan S; Kurabayashi, Masahiko

2013-01-01

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the (125)I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.

A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

PubMed Central

Syamsunarno, Mas Rizky A. A.; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; YoshitoTsushima; Endo, Keigo; Hotamisligil, Gökhan S.; Kurabayashi, Masahiko

2013-01-01

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the 125I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis. PMID:24244493

High-resolution vascular tissue characterization in mice using 55MHz ultrasound hybrid imaging.

PubMed

Mahmoud, Ahmed M; Sandoval, Cesar; Teng, Bunyen; Schnermann, Jurgen B; Martin, Karen H; Mustafa, S Jamal; Mukdadi, Osama M

2013-03-01

Ultrasound and Duplex ultrasonography in particular are routinely used to diagnose cardiovascular disease (CVD), which is the leading cause of morbidity and mortality worldwide. However, these techniques may not be able to characterize vascular tissue compositional changes due to CVD. This work describes an ultrasound-based hybrid imaging technique that can be used for vascular tissue characterization and the diagnosis of atherosclerosis. Ultrasound radiofrequency (RF) data were acquired and processed in time, frequency, and wavelet domains to extract six parameters including time integrated backscatter (T(IB)), time variance (T(var)), time entropy (T(E)), frequency integrated backscatter (F(IB)), wavelet root mean square value (W(rms)), and wavelet integrated backscatter (W(IB)). Each parameter was used to reconstruct an image co-registered to morphological B-scan. The combined set of hybrid images were used to characterize vascular tissue in vitro and in vivo using three mouse models including control (C57BL/6), and atherosclerotic apolipoprotein E-knockout (APOE-KO) and APOE/A(1) adenosine receptor double knockout (DKO) mice. The technique was tested using high-frequency ultrasound including single-element (center frequency=55 MHz) and commercial array (center frequency=40 MHz) systems providing superior spatial resolutions of 24 μm and 40 μm, respectively. Atherosclerotic vascular lesions in the APOE-KO mouse exhibited the highest values (contrast) of -10.11±1.92 dB, -12.13±2.13 dB, -7.54±1.45 dB, -5.10±1.06 dB, -5.25±0.94 dB, and -10.23±2.12 dB in T(IB), T(var), T(E), F(IB), W(rms), W(IB) hybrid images (n=10, p<0.05), respectively. Control segments of normal vascular tissue showed the lowest values of -20.20±2.71 dB, -22.54±4.54 dB, -14.94±2.05 dB, -9.64±1.34 dB, -10.20±1.27 dB, and -19.36±3.24 dB in same hybrid images (n=6, p<0.05). Results from both histology and optical images showed good agreement with ultrasound findings within a maximum error of 3.6% in lesion estimation. This study demonstrated the feasibility of a high-resolution hybrid imaging technique to diagnose atherosclerosis and characterize plaque components in mouse. In the future, it can be easily implemented on commercial ultrasound systems and eventually translated into clinics as a screening tool for atherosclerosis and the assessment of vulnerable plaques. Copyright © 2012 Elsevier B.V. All rights reserved.

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity.

PubMed

Dubielecka, Patrycja M; Ladwein, Kathrin I; Xiong, Xiaoling; Migeotte, Isabelle; Chorzalska, Anna; Anderson, Kathryn V; Sawicki, Janet A; Rottner, Klemens; Stradal, Theresia E; Kotula, Leszek

2011-04-26

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

PubMed Central

Dubielecka, Patrycja M.; Ladwein, Kathrin I.; Xiong, Xiaoling; Migeotte, Isabelle; Chorzalska, Anna; Anderson, Kathryn V.; Sawicki, Janet A.; Rottner, Klemens; Stradal, Theresia E.; Kotula, Leszek

2011-01-01

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function. PMID:21482783

12 CFR 3.135 - Guarantees and credit derivatives: double default treatment.

Code of Federal Regulations, 2014 CFR

2014-01-01

... to the following formula: KDD = Ko × (0.15 + 160 × PDg), Where: (1) ER11OC13.048 (2) PDg = PD of the... according to the appropriate formula for (R) provided in Table 1 in § 3.131, with PD equal to PDo. (6) b... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Guarantees and credit derivatives: double...

Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Furuie, Sumie; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Yamamura, Ken-Ichi; Kobayashi, Keiko

2012-11-01

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight. Copyright © 2012 Elsevier Inc. All rights reserved.

Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli.

PubMed

Hu, Fengqi; Ding, Guohua; Zhang, Zhiyong; Gatto, Louis A; Hawgood, Samuel; Poulain, Francis R; Cooney, Robert N; Wang, Guirong

2016-01-01

To investigate the effects of surfactant proteins A and D (SP-A and SP-D, respectively) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI, as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared with control, the level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than in infected WT mice 24 and 48 h post-infection. The basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher than in WT mice. The phosphorylated p38 level was elevated in the kidney of WT mice post infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI. © The Author(s) 2015.

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

PubMed Central

Vereecke, Lars; Mc Guire, Conor; Sze, Mozes; Schuijs, Martijn J.; Willart, Monique; Itati Ibañez, Lorena; Hammad, Hamida; Lambrecht, Bart N.; Beyaert, Rudi; Saelens, Xavier; van Loo, Geert

2016-01-01

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection. PMID:26815999

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice.

PubMed

Zhang, Hong-Mei; Chen, Shao-Rui; Matsui, Minoru; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2006-03-01

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs.

PubMed

Stanley-Hasnain, Shanna; Hauck, Ludger; Grothe, Daniela; Aschar-Sobbi, Roozbeh; Beca, Sanja; Butany, Jagdish; Backx, Peter H; Mak, Tak W; Billia, Filio

2017-01-01

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21 Cip1 and p27 Kip1 . Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.

Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

PubMed

Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

2015-07-01

Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete. Copyright © 2015 Elsevier GmbH. All rights reserved.

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Running promotes wakefulness and increases cataplexy in orexin knockout mice.

PubMed

España, Rodrigo A; McCormack, Sarah L; Mochizuki, Takatoshi; Scammell, Thomas E

2007-11-01

People with narcolepsy and mice lacking orexin/hypocretin have disrupted sleep/wake behavior and reduced physical activity. Our objective was to identify physiologic mechanisms through which orexin deficiency reduces locomotor activity. We examined spontaneous wheel running activity and its relationship to sleep/wake behavior in wild type (WT) and orexin knockout (KO) mice. Additionally, given that physical activity promotes alertness, we also studied whether orexin deficiency reduces the wake-promoting effects of exercise. Orexin KO mice ran 42% less than WT mice. Their ability to run appeared normal as they initiated running as often as WT mice and ran at normal speeds. However, their running bouts were considerably shorter, and they often had cataplexy or quick transitions into sleep after running. Wheel running increased the total amount of wakefulness in WT and orexin KO mice similarly, however, KO mice continued to have moderately fragmented sleep/wake behavior. Wheel running also doubled the amount of cataplexy by increasing the probability of transitioning into cataplexy. Orexin KO mice run significantly less than normal, likely due to sleepiness, imminent cataplexy, or a reduced motivation to run. Orexin is not required for the wake-promoting effects of wheel running given that both WT and KO mice had similar increases in wakefulness with running wheels. In addition, the clear increase in cataplexy with wheel running suggests the possibility that positive emotions or reward can trigger murine cataplexy, similar to that seen in people and dogs with narcolepsy.

Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

PubMed Central

Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

2014-01-01

Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

Activity-dependent extracellular K+ accumulation in rat optic nerve: the role of glial and axonal Na+ pumps

PubMed Central

Ransom, Christopher B; Ransom, Bruce R; Sontheimer, Harald

2000-01-01

We measured activity-dependent changes in [K+]o with K+-selective microelectrodes in adult rat optic nerve, a CNS white matter tract, to investigate the factors responsible for post-stimulus recovery of [K+]o.Post-stimulus recovery of [K+]o followed a double-exponential time course with an initial, fast time constant, τfast, of 0.9 ± 0.2 s (mean ±s.d.) and a later, slow time constant, τslow, of 4.2 ± 1 s following a 1 s, 100 Hz stimulus. τfast, but not τslow, decreased with increasing activity-dependent rises in [K+]o. τslow, but not τfast, increased with increasing stimulus duration.Post-stimulus recovery of [K+]o was temperature sensitive. The apparent temperature coefficients (Q10, 27–37°C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively.Post-stimulus recovery of [K+]o was sensitive to Na+ pump inhibition with 50 μM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 μM strophanthidin increased τfast and τslow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post-stimulus recovery of [K+]o.Post-stimulus recovery of [K+]o was minimally affected by the K+ channel blocker Ba2+ (0.2 mm). Following a 10 s, 100 Hz stimulus, 0.2 mm Ba2+ increased τfast and τslow by 24 and 18%, respectively.Stimulated increases in [K+]o were followed by undershoots of [K+]o. Post-stimulus undershoot amplitude increased with stimulus duration but was independent of the peak preceding [K+]o increase.These observations imply that two distinct processes contribute to post-stimulus recovery of [K+]o in central white matter. The results are compatible with a model of K+ removal that attributes the fast, initial phase of K+ removal to K+ uptake by glial Na+ pumps and the slower, sustained decline to K+ uptake via axonal Na+ pumps. PMID:10713967

Expression at the imprinted dlk1-gtl2 locus is regulated by proneural genes in the developing telencephalon.

PubMed

Seibt, Julie; Armant, Olivier; Le Digarcher, Anne; Castro, Diogo; Ramesh, Vidya; Journot, Laurent; Guillemot, François; Vanderhaeghen, Pierre; Bouschet, Tristan

2012-01-01

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.

Expression at the Imprinted Dlk1-Gtl2 Locus Is Regulated by Proneural Genes in the Developing Telencephalon

PubMed Central

Seibt, Julie; Armant, Olivier; Le Digarcher, Anne; Castro, Diogo; Ramesh, Vidya; Journot, Laurent; Guillemot, François; Vanderhaeghen, Pierre; Bouschet, Tristan

2012-01-01

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon. PMID:23139813

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

PubMed

Bossi, Simone; Musante, Ilaria; Bonfiglio, Tommaso; Bonifacino, Tiziana; Emionite, Laura; Cerminara, Maria; Cervetto, Chiara; Marcoli, Manuela; Bonanno, Giambattista; Ravazzolo, Roberto; Pittaluga, Anna; Puliti, Aldamaria

2018-01-01

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1 crv4/crv4 ) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1 crv4 and Grm5 ko mice to generate double mutants (Grm1 crv4/crv4 Grm5 ko/ko ) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1 crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1 crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1 crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1 crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Increase of Total Nephron Albumin Filtration and Reabsorption in Diabetic Nephropathy.

PubMed

Mori, Keita P; Yokoi, Hideki; Kasahara, Masato; Imamaki, Hirotaka; Ishii, Akira; Kuwabara, Takashige; Koga, Kenichi; Kato, Yukiko; Toda, Naohiro; Ohno, Shoko; Kuwahara, Koichiro; Endo, Tomomi; Nakao, Kazuwa; Yanagita, Motoko; Mukoyama, Masashi; Mori, Kiyoshi

2017-01-01

The amount of albumin filtered through the glomeruli and reabsorbed at the proximal tubules in normal and in diabetic kidneys is debated. The megalin/cubilin complex mediates protein reabsorption, but genetic knockout of megalin is perinatally lethal. To overcome current technical problems, we generated a drug-inducible megalin-knockout mouse line, megalin(lox/lox);Ndrg1-CreER T2 (iMegKO), in which megalin expression can be shut off at any time by administration of tamoxifen (Tam). Tam administration in adult iMegKO mice decreased the expression of renal megalin protein by 92% compared with that in wild-type C57BL/6J mice and almost completely abrogated renal reabsorption of intravenously injected retinol-binding protein. Furthermore, urinary albumin excretion increased to 175 μg/d (0.46 mg albumin/mg creatinine) in Tam-treated iMegKO mice, suggesting that this was the amount of total nephron albumin filtration. By comparing Tam-treated, streptozotocin-induced diabetic iMegKO mice with Tam-treated nondiabetic iMegKO mice, we estimated that the development of diabetes led to a 1.9-fold increase in total nephron albumin filtration, a 1.8-fold increase in reabsorption, and a significant reduction in reabsorption efficiency (86% efficiency versus 96% efficiency in nondiabetic mice). Insulin treatment normalized these abnormalities. Akita;iMegKO mice, another model of type 1 diabetes, showed equivalent results. Finally, nondiabetic iMegKO mice had a glomerular sieving coefficient of albumin of 1.7×10 -5 , which approximately doubled in diabetic iMegKO mice. This study reveals actual values and changes of albumin filtration and reabsorption in early diabetic nephropathy in mice, bringing new insights to our understanding of renal albumin dynamics associated with the hyperfiltration status of diabetic nephropathy. Copyright © 2016 by the American Society of Nephrology.

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellularmore » response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.« less

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

PubMed Central

Ruusuvuori, Eva; Huebner, Antje K; Kirilkin, Ilya; Yukin, Alexey Y; Blaesse, Peter; Helmy, Mohamed; Jung Kang, Hyo; El Muayed, Malek; Christopher Hennings, J; Voipio, Juha; Šestan, Nenad; Hübner, Christian A; Kaila, Kai

2013-01-01

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13–14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13–P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS. PMID:23881097

Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice.

PubMed

Westerterp, Marit; Murphy, Andrew J; Wang, Mi; Pagler, Tamara A; Vengrenyuk, Yuliya; Kappus, Mojdeh S; Gorman, Darren J; Nagareddy, Prabhakara R; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S; Welch, Carrie; Fisher, Edward A; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R

2013-05-24

Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. To assess the role of macrophage cholesterol efflux pathways in atherogenesis. We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice.

PubMed

Markham, Bruce E; Kernodle, Stace; Nemzek, Jean; Wilkinson, John E; Sigler, Robert

2015-01-01

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone's effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice

PubMed Central

Markham, Bruce E.; Kernodle, Stace; Nemzek, Jean; Wilkinson, John E.; Sigler, Robert

2015-01-01

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone’s effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients. PMID:26248188

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa.

PubMed

Shankar, Sharmila; Srivastava, Rakesh K

2007-06-01

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.

ID2 collaborates with ID3 to suppress iNKT and innate-like tumors1

PubMed Central

Li, Jia; Roy, Sumedha; Kim, Young-Mi; Li, Shibo; Zhang, Baojun; Love, Cassandra; Reddy, Anupama; Rajagopalan, Deepthi; Dave, Sandeep; Diehl, Anna Mae; Zhuang, Yuan

2017-01-01

Inhibitor of DNA binding (ID) proteins, including ID1-4, are transcriptional regulators involved in promoting cell proliferation and survival in various cell types. Although upregulation of Id proteins has been associated with a broad spectrum of tumors, recent studies have identified that ID3 plays a tumor suppressor role in the development of Burkitt’s lymphoma in humans and Hepatosplenic T cell lymphomas in mice. Here, we report rapid lymphoma development in Id2/Id3 double knockout (L-DKO) mice caused by unchecked expansion of either invariant Natural Killer T (iNKT) cells, or a unique subset of innate-like, CD1d-independent T cells. These populations started expansion in neonatal mice and, upon malignant transformation, caused fatality at age between 3–11 months. The malignant cells also gave rise to lymphomas upon transfer to Rag-deficient and wild-type hosts, reaffirming their inherent tumorigenic potential. Microarray analysis revealed a significantly modified program in these neonatal iNKT cells that ultimately led to their malignant transformation. The lymphoma cells demonstrated chromosome instability, along with upregulation of several different signaling pathways, including the cytokine-cytokine receptor interaction pathway, which can promote their expansion and migration. Dysregulation of genes with reported driver mutations and the NF-kB pathway were found to be shared between L-DKO lymphomas and human NKT tumors. Our work identifies a distinct premalignant state and multiple tumoriogenic pathways caused by loss function of ID2 and ID3. Thus, conditional deletion of Id2 and Id3 in developing T cells establishes a unique animal model for iNKT and relevant innate-like lymphomas. PMID:28258199

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

PubMed

Church, Jarrod E; Trieu, Jennifer; Chee, Annabel; Naim, Timur; Gehrig, Stefan M; Lamon, Séverine; Angelini, Corrado; Russell, Aaron P; Lynch, Gordon S

2014-04-01

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Development of mice without Cip/Kip CDK inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu

2012-10-19

Highlights: Black-Right-Pointing-Pointer Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. Black-Right-Pointing-Pointer Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. Black-Right-Pointing-Pointer CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largelymore » unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G{sub 0} to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage).« less

TNF-α and Temporal Changes in Sleep Architecture in Mice Exposed to Sleep Fragmentation

PubMed Central

2012-01-01

TNF-α plays critical roles in host-defense, sleep-wake regulation, and the pathogenesis of various disorders. Increases in the concentration of circulating TNF-α after either sleep deprivation or sleep fragmentation (SF) appear to underlie excessive daytime sleepiness in patients with sleep apnea (OSA). Following baseline recordings, mice were subjected to 15 days of SF (daily for 12 h/day from 07.00 h to 19.00 h), and sleep parameters were recorded on days1, 7 and 15. Sleep architecture and sleep propensity were assessed in both C57BL/6J and in TNF-α double receptor KO mice (TNFR KO). To further confirm the role of TNF-α, we also assessed the effect of treatment with a TNF- α neutralizing antibody in C57BL/6J mice. SF was not associated with major changes in global sleep architecture in C57BL/6J and TNFR KO mice. TNFR KO mice showed higher baseline SWS delta power. Further, following 15 days of SF, mice injected with TNF-α neutralizing antibody and TNFR KO mice showed increased EEG SWS activity. However, SWS latency, indicative of increased propensity to sleep, was only decreased in C57BL/6J, and was unaffected in TNFR KO mice as well as in C57BL/6J mice exposed to SF but treated with TNF-α neutralizing antibody. Taken together, our findings show that the excessive sleepiness incurred by recurrent arousals during sleep may be due to activation of TNF-alpha-dependent inflammatory pathways, despite the presence of preserved sleep duration and global sleep architecture. PMID:23029133

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

PubMed

Alvarez, Marta B; Childress, Paul; Philip, Binu K; Gerard-O'Riley, Rita; Hanlon, Michael; Herbert, Brittney-Shea; Robling, Alexander G; Pavalko, Fredrick M; Bidwell, Joseph P

2012-05-01

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. Copyright © 2011 Wiley Periodicals, Inc.

Running Promotes Wakefulness and Increases Cataplexy in Orexin Knockout Mice

PubMed Central

España, Rodrigo A.; McCormack, Sarah L.; Mochizuki, Takatoshi; Scammell, Thomas E.

2007-01-01

Study Objective: People with narcolepsy and mice lacking orexin/hypocretin have disrupted sleep/wake behavior and reduced physical activity. Our objective was to identify physiologic mechanisms through which orexin deficiency reduces locomotor activity. Design: We examined spontaneous wheel running activity and its relationship to sleep/wake behavior in wild type (WT) and orexin knockout (KO) mice. Additionally, given that physical activity promotes alertness, we also studied whether orexin deficiency reduces the wake-promoting effects of exercise. Measurements and Results: Orexin KO mice ran 42% less than WT mice. Their ability to run appeared normal as they initiated running as often as WT mice and ran at normal speeds. However, their running bouts were considerably shorter, and they often had cataplexy or quick transitions into sleep after running. Wheel running increased the total amount of wakefulness in WT and orexin KO mice similarly, however, KO mice continued to have moderately fragmented sleep/wake behavior. Wheel running also doubled the amount of cataplexy by increasing the probability of transitioning into cataplexy. Conclusions: Orexin KO mice run significantly less than normal, likely due to sleepiness, imminent cataplexy, or a reduced motivation to run. Orexin is not required for the wake-promoting effects of wheel running given that both WT and KO mice had similar increases in wakefulness with running wheels. In addition, the clear increase in cataplexy with wheel running suggests the possibility that positive emotions or reward can trigger murine cataplexy, similar to that seen in people and dogs with narcolepsy. Citation: España RA; McCormack SL; Mochizuki T; Scammell TE. Running promotes wakefulness and increases cataplexy in orexin knockout mice. SLEEP 2007;30(11):1417-1425. PMID:18041476

Augmentation of the noradrenergic system in alpha-2 adrenergic receptor deficient mice: anatomical changes associated with enhanced fear memory.

PubMed

Davies, M Frances; Tsui, Janet Y; Flannery, Judy A; Li, Xiangqi; DeLorey, Timothy M; Hoffman, Brian B

2003-10-03

We have investigated sensitivity to the conditioned fear procedure of mice is influenced by the genetic deletion of alpha2A adrenoceptors (ARs). We observed a heightened freezing response in the discrete cue memory test in alpha2A AR knockout (alpha2A AR KO) mice and in D79N mice, a transgenic mouse strain with functionally impaired alpha2A ARs. No significant differences in contextual memory were observed between control and alpha2A AR KO or D79N mice suggesting a minimal role for the noradrenergic system in contextual memory. We speculated that the increased freezing response of the alpha2A AR KO and D79N mice in the discrete cue setting was due to increased release of norepinephrine evoked by the unconditioned footshock stimulus. In alpha2A AR KO mice we measured a doubling in the number of noradrenergic neurons in the locus coeruleus (LC) and a large increase in the cell volume of tyrosine hydroxylase positive neurons, likely due to selective preservation of large, multipolar neurons in the subcoeruleus. Hyperplasia of the noradrenergic neurons in the nucleus tractus solitarius, A5 and A7, was also observed. Alpha2A AR KO mice exhibit greater c-Fos expression in the LC compared to wild type mice suggesting that the LC neurons in the alpha2A AR KO mice were spontaneously more active. This study suggests that alpha2A ARs are involved in the development of the central noradrenergic system and raises the possibility that alterations in alpha2A AR expression may contribute to variations in fear and stress responses.

Functional characterization of Bombyx mori nucleopolyhedrovirus mutant lacking late expression factor 9.

PubMed

Zhang, Y; Shi, Y; Yu, H; Li, J; Quan, Y; Shu, T; Nie, Z; Zhang, Y; Yu, W

Baculoviridae is a family of invertebrate viruses with large double-stranded DNA genomes. Proteins encoded by some late expression factor (lef ) genes are involved in the regulation of viral gene expression. Lef-9 is one of four transcription-specific Lefs, which are components of the virus-encoded RNA polymerase, and can initiate and transcribe late and very late genes. As a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus (BmNPV), Lef-9 may be involved in the regulation of viral propagation. However, the underlying mechanism remains unclear. To determine the role of lef-9 in baculovirus infection, lef-9-knockout virus (lef-9-KO-Bacmid virus) was constructed using the Red recombination system, and the Bac-to-Bac system was used to prepare lef-9-repaired virus (lef-9-Re-Bacmid virus). The lef-9-KO virus did not produce infectious viruses or show infection activity, while the lef-9-repaired virus recovered both. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the transcription levels in wild-type-Bacmid, lef-9-KO-Bacmid, and lef-9-Re-Bacmid viruses showed that the lef-9-KO bacmid had little effect on viral genome replication. However, the transcription levels of the early and late viral genes, lef-3, ie-1, vp39, and p10, were significantly lower in BmN cells transfected with lef-9-KO-Bacmids than in the controls. Electron microscopy showed no visible enveloped virions in cells transfected with lef-9-KO-Bacmids, while many mature virions in cells transfected with lef-9-Re-Bacmid and wt-Bacmid were present. Thus, lef-9 was not essential for viral genome replication, but significantly affected viral gene transcription and expression in all periods of cell life cycle.

The roles of sex and serotonin transporter levels in age- and stress-related emotionality in mice.

PubMed

Joeyen-Waldorf, Jennifer; Edgar, Nicole; Sibille, Etienne

2009-08-25

Mood disorders are influenced by genetic make-up and differentially affect men and women. The s/l promoter polymorphism in the serotonin transporter (SERT) gene moderates both trait emotion and the vulnerability to develop depressive states in humans. Similarly, male mice lacking SERT (Knockout/KO) display an elevated emotionality phenotype. We now report that the SERT-KO phenotype is maintained throughout late-adulthood, and that female KO mice develop a larger emotionality phenotype with increasing age. Thus, to test the hypothesis that these findings reflected a putative sexual dimorphism in SERT-mediated modulation of emotionality, we submitted adult male and female wild-type, heterozygous (HZ) and KO mice to unpredictable chronic mild stress (UCMS) and assessed behavioral changes. In males, the elevated SERT-KO emotion-related behavior converged with other groups after UCMS. Conversely, female SERT-KO displayed a normal non-stressed baseline, but highest UCMS-induced emotionality. SERT-HZ displayed variable and intermediate phenotypes in both experiments. Thus, consistent results across different biological modalities (age, stress) revealed a high contribution of SERT genotype for baseline "trait" emotionality in males, and low contribution for females. In contrast, age-correlated and stress-induced behavioral changes resulted in a high SERT genotype-mediated behavioral variance in females, but low in males. This suggests that high emotionality states associated with low SERT were differentially achieved in males (high baseline/trait) compared to females (increased vulnerability to develop high emotionality). This sex-by-SERT double dissociation provides a framework to investigate molecular substrates of emotionality regulation in concert with serotonin function and may contribute to the sexually dimorphic features of mood disorders.

Genomic deletion of GIT2 induces a premature age-related thymic dysfunction and systemic immune system disruption

PubMed Central

Siddiqui, Sana; Lustig, Ana; Carter, Arnell; Sankar, Mathavi; Daimon, Caitlin M.; Premont, Richard T.; Etienne, Harmonie; van Gastel, Jaana; Azmi, Abdelkrim; Janssens, Jonathan; Becker, Kevin G.; Zhang, Yongqing; Wood, William; Lehrmann, Elin; Martin, James G.; Martin, Bronwen; Taub, Dennis D.; Maudsley, Stuart

2017-01-01

Recent research has proposed that GIT2 (G protein-coupled receptor kinase interacting protein 2) acts as an integrator of the aging process through regulation of ‘neurometabolic’ integrity. One of the commonly accepted hallmarks of the aging process is thymic involution. At a relatively young age, 12 months old, GIT2−/− mice present a prematurely distorted thymic structure and dysfunction compared to age-matched 12 month-old wild-type control (C57BL/6) mice. Disruption of thymic structure in GIT2−/− (GIT2KO) mice was associated with a significant reduction in the expression of the cortical thymic marker, Troma-I (cytokeratin 8). Double positive (CD4+CD8+) and single positive CD4+ T cells were also markedly reduced in 12 month-old GIT2KO mice compared to age-matched control wild-type mice. Coincident with this premature thymic disruption in GIT2KO mice was the unique generation of a novel cervical ‘organ’, i.e. ‘parathymic lobes’. These novel organs did not exhibit classical peripheral lymph node-like characteristics but expressed high levels of T cell progenitors that were reflexively reduced in GIT2KO thymi. Using signaling pathway analysis of GIT2KO thymus and parathymic lobe transcriptomic data we found that the molecular signaling functions lost in the dysfunctional GIT2KO thymus were selectively reinstated in the novel parathymic lobe – suggestive of a compensatory effect for the premature thymic disruption. Broader inspection of high-dimensionality transcriptomic data from GIT2KO lymph nodes, spleen, thymus and parathymic lobes revealed a systemic alteration of multiple proteins (Dbp, Tef, Per1, Per2, Fbxl3, Ddit4, Sin3a) involved in the multidimensional control of cell cycle clock regulation, cell senescence, cellular metabolism and DNA damage. Altered cell clock regulation across both immune and non-immune tissues therefore may be responsible for the premature ‘aging’ phenotype of GIT2KO mice. PMID:28260693

Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

PubMed

Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

2014-06-01

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Enhancement of pump absorption efficiency by bending and twisting of double clad rare earth doped fibers (Conference Presentation)

NASA Astrophysics Data System (ADS)

Koška, Pavel; Peterka, Pavel; Doya, Valérie; Aubrecht, Jan; Kasik, Ivan; Podrazký, Ondřej

2017-05-01

High-power operation of fiber lasers was enabled by the invention of cladding-pumping in a double-clad fiber structure. Because of existence of so called skew rays in the inner clad of the fiber, pump absorption saturates along the fiber and pumping becomes inefficient. First studies of pump absorption efficiency enhancement were focused on fibers with broken circular symmetry of inner cladding eliminating skew rays [1,2]. Later, techniques of unconventional fiber coiling were proposed [3]. However, theoretical studies were limited to the assumption of a straight fiber. Even recently, the rigorous model accounting for fiber bending and twisting was described [4-6]. It was found that bending of the fiber influences modal spectra of the pump radiation and twisting provides quite efficient mode-scrambling. These effects in a synergic manner significantly enhances pump absorption rate in double clad fibers and improves laser system efficiency. In our contribution we review results of numerical modelling of pump absorption in various types of double-clad fibers, e.g., with cross section shape of hexagon, stadium, and circle; two-fiber bundle (so-called GTWave fiber structure) a panda fibers are also analyzed. We investigate pump field modal spectra evolution in hexagonally shaped fiber in straight, bended, and simultaneously bended and twisted fiber which brings new quality to understanding of the mode-scrambling and pump absorption enhancement. Finally, we evaluate the impact of enhanced pump absorption on signal gain in the fiber. These results can have practical impact in construction of fiber lasers: with pump absorption efficiency optimized by our new model (the other models did not take into account fiber twist), the double-clad fiber of shorter length can be used in the fiber lasers and amplifiers. In such a way the harmful influence of background losses and nonlinear effects can be minimized. [1] Doya, V., Legrand, O., Mortessagne, F., "Optimized absorption in a chaotic double-clad fiber amplifier," Opt. Lett., vol. 26, no. 12, pp. 872-874, (2001). [2] Kouznetsov, D., Moloney, J. V., "Efficiency of pump absorption in double-clad fiber amplifiers. II. Broken circular symmetry," J. Opt. Soc. Am. B, vol. 19, no. 6, pp. 1259-1263, June 2002. [3] Li, Y., Jackson, S. D., Fleming, S., "High absorption and low splice loss properties of hexagonal double-clad fiber," IEEE Photonics Technol. Lett., vol 16, no. 11, pp. 2502-2504, Nov. 2004. [4] Ko\\vska, P. and Peterka, P., "Numerical analysis of pump propagation and absorption in specially tailored double-clad rare-earth doped fiber," Optical and Quantum Electronics, vol. 47, no. 9, pp. 3181-3191 (2015). [5] Ko\\vska, P., Peterka, P., and Doya, V., "Numerical modeling of pump absorption in coiled and twisted double-clad fibers," IEEE J. Sel. Top. Quantum Electron., vol. 22, no. 2 (2016). [6] Ko\\vska, P., Peterka, P., Aubrecht, J., Podrazký, O., Todorov, F., Becker, M., Baravets, Y., Honzátko, P., and Kašík, I., "Enhanced pump absorption efficiency in coiled and twisted double-clad thulium-doped fibers," Opt. Express, vol. 24, no. 1, pp. 102-107 (2016).

Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver.

PubMed

Hinrichs, Arne; Kessler, Barbara; Kurome, Mayuko; Blutke, Andreas; Kemter, Elisabeth; Bernau, Maren; Scholz, Armin M; Rathkolb, Birgit; Renner, Simone; Bultmann, Sebastian; Leonhardt, Heinrich; de Angelis, Martin Hrabĕ; Nagashima, Hiroshi; Hoeflich, Andreas; Blum, Werner F; Bidlingmaier, Martin; Wanke, Rüdiger; Dahlhoff, Maik; Wolf, Eckhard

2018-05-01

Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue. GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2. GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Deficiency of endothelial CXCR4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice.

PubMed

Noels, Heidi; Zhou, Baixue; Tilstam, Pathricia V; Theelen, Wendy; Li, Xiaofeng; Pawig, Lukas; Schmitz, Corinna; Akhtar, Shamima; Simsekyilmaz, Sakine; Shagdarsuren, Erdenechimeg; Schober, Andreas; Adams, Ralf H; Bernhagen, Jürgen; Liehn, Elisa A; Döring, Yvonne; Weber, Christian

2014-06-01

The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. β-Galactosidase staining using bone marrow x kinase (Bmx)-CreER(T2) reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER(T2+) compared with Bmx-CreER(T2-) Cxcr4-floxed apolipoprotein E-deficient (Apoe(-/-)) mice (referred to as Cxcr4(EC-KO)ApoE(-/-) and Cxcr4(EC-WT) ApoE(-/-), respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4(EC-KO)ApoE(-/-) mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4(EC-KO)ApoE(-/-) carotids compared with Cxcr4(EC-WT)ApoE(-/-) controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1(+)Flk1(+)Cd31(+) and of Lin(-)Sca1(+) progenitors in Cxcr4(EC-KO) ApoE(-/-) mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4(EC-KO)ApoE(-/-) mice. Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1(+)Flk1(+)Cd31(+) cells, often referred to as circulating endothelial progenitor cells. © 2014 American Heart Association, Inc.

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.

PubMed

Wang, Hao; Jurado, Kellie A; Wu, Xiaolin; Shun, Ming-Chieh; Li, Xiang; Ferris, Andrea L; Smith, Steven J; Patel, Pratiq A; Fuchs, James R; Cherepanov, Peter; Kvaratskhelia, Mamuka; Hughes, Stephen H; Engelman, Alan

2012-12-01

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

PubMed

Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

2016-04-01

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

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

PubMed Central

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 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 abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel. PMID:29201008

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

PubMed

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 < 0.05). The presence of the LRAP Tg did not improve the phenotype of M180 Tg /CTRNC Tg /KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle.

PubMed

Kitazawa, Takio; Hirama, Ryuichi; Masunaga, Kozue; Nakamura, Tatsuro; Asakawa, Koichi; Cao, Jinshan; Teraoka, Hiroki; Unno, Toshihiro; Komori, Sei-ichi; Yamada, Masahisa; Wess, Jürgen; Taneike, Tetsuro

2008-06-01

Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via reverse transcription polymerase chain reaction. Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the concentration-response curve was shifted to the right and downward compared with that for the corresponding wild-type mice. On the other hand, uterine strips isolated from M3KO and M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor. Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor activation enhanced M3 receptor-mediated contractions in the mouse uterus.

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca(2+) and proton flux dysregulation.

PubMed

Domenighetti, Andrea A; Danes, Vennetia R; Curl, Claire L; Favaloro, Jennifer M; Proietto, Joseph; Delbridge, Lea M D

2010-04-01

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, >95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca(2+) current (I(Ca)) and Na(+)-Ca(2+) exchanger (NCX) fluxes, Na(+)-H(+) exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0+/-0.4% vs. 6.2+/-0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I(Ca) density was decreased (-2.80+/-0.29 vs. -5.30+/-0.70 pA/pF), and mean I(NCX) was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H(+) flux in response to NH(4)Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca(2+) and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

A practical theoretical formalism for atomic multielectron processes: direct multiple ionization by a single auger decay or by impact of a single electron or photon

NASA Astrophysics Data System (ADS)

Liu, Pengfei; Zeng, Jiaolong; Yuan, Jianmin

2018-04-01

Multiple electron processes occur widely in atoms, molecules, clusters, and condensed matters when they are interacting with energetic particles or intense laser fields. Direct multielectron processes (DMEP) are the most complicated among the general multiple electron processes and are the most difficult to describe theoretically. In this work, a unified and accurate theoretical formalism is proposed on the DMEP of atoms including the multiple auger decay and multiple ionization by an impact of a single electron or a single photon based on the atomic collision theory described by a correlated many-body Green's function. Such a practical treatment is made possible by taking consideration of the different coherence features of the atoms (matter waves) in the initial and final states. We first explain how the coherence characteristics of the ejected continuum electrons is largely destructed, by taking the electron impact direct double ionization process as an example. The direct double ionization process is completely different from the single ionization where the complete interference can be maintained. The detailed expressions are obtained for the energy correlations among the continuum electrons and energy resolved differential and integral cross sections according to the separation of knock-out (KO) and shake-off (SO) mechanisms for the electron impact direct double ionization, direct double and triple auger decay, and double and triple photoionization (TPI) processes. Extension to higher order DMEP than triple ionization is straight forward by adding contributions of the following KO and SO processes. The approach is applied to investigate the electron impact double ionization processes of C+, N+, and O+, the direct double and triple auger decay of the K-shell excited states of C+ 1s2{s}22{p}2{}2D and {}2P, and the double and TPI of lithium. Comparisons with the experimental and other theoretical investigations wherever available in the literature show that our theoretical formalism is accurate and effective in treating the atomic multielectron processes.

Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes

PubMed Central

Garcia-Alvarez, Gisela; Shetty, Mahesh S.; Lu, Bo; Yap, Kenrick An Fu; Oh-Hora, Masatsugu; Sajikumar, Sreedharan; Bichler, Zoë; Fivaz, Marc

2015-01-01

Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca2+ channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories. PMID:26236206

Endothelin receptor antagonist macitentan or deletion of mouse mast cell protease 4 delays lesion development in atherosclerotic mice.

PubMed

Houde, Martin; Desbiens, Louisane; Schwertani, Adel; Pejler, Gunnar; Iglarz, Marc; D'Orléans-Juste, Pedro

2016-08-15

To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52weeks. Macitentan (30mg/kg/day), a dual ETA/ETB receptor antagonist, was given orally for 6weeks (27week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Masson's trichrome stain and α-smooth muscle actin labeling. Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent. Copyright © 2016 Elsevier Inc. All rights reserved.

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice.

PubMed

Seki, Ekihiro; Tsutsui, Hiroko; Tsuji, Noriko M; Hayashi, Nobuki; Adachi, Keishi; Nakano, Hiroki; Futatsugi-Yumikura, Shizue; Takeuchi, Osamu; Hoshino, Katsuaki; Akira, Shizuo; Fujimoto, Jiro; Nakanishi, Kenji

2002-10-01

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.

The Interaction between Regulatory T Cells and NKT Cells in the Liver: A CD1d Bridge Links Innate and Adaptive Immunity

PubMed Central

Webb, Tonya J.; Potter, James P.; Li, Zhiping

2011-01-01

Background/Aims Regulatory T cells (Tregs) and natural killer T (NKT) cells are two distinct lymphocyte subsets that independently regulate hepatic adaptive and innate immunity, respectively. In the current study, we examine the interaction between Tregs and NKT cells to understand the mechanisms of cross immune regulation by these cells. Methods The frequency and function of Tregs were evaluated in wild type and NKT cell deficient (CD1dko) mice. In vitro lymphocyte proliferation and apoptosis assays were performed with NKT cells co-cultured with Tregs. The ability of Tregs to inhibit NKT cells in vivo was examined by adoptive transfer of Tregs in a model of NKT cell mediated hepatitis. Results CD1dko mice have a significant reduction in hepatic Tregs. Although, the Tregs from CD1dko mice remain functional and can suppress conventional T cells, their ability to suppress activation induced NKT cell proliferation and to promote NKT cell apoptosis is greatly diminished. These effects are CD1d dependent and require cell to cell contact. Adoptive transfer of Tregs inhibits NKT cell-mediated liver injury. Conclusions NKT cells promote Tregs, and Tregs inhibit NKT cells in a CD1d dependent manner requiring cell to cell contact. These cross-talk immune regulations provide a linkage between innate and adaptive immunity. PMID:22073248

L-2-oxothiazolidine-4-carboxylic acid attenuates oxidative stress and inflammation in retinal pigment epithelium

PubMed Central

Promsote, Wanwisa; Veeranan-Karmegam, Rajalakshmi; Ananth, Sudha; Shen, Defen; Chan, Chi-Chao; Lambert, Nevin A.; Ganapathy, Vadivel

2014-01-01

Purpose Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells is central to the pathogenesis of age-related macular degeneration (AMD). Thus, developing novel strategies to protect these cells is important. We reported previously on the robust antioxidant and therefore cell-protective effects of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) in cultured human RPE cells. New reports citing a novel anti-inflammatory role for OTC in addition to the known glutathione-stimulating and antioxidant properties emerged recently; however, this role has not been evaluated in RPE cells or in intact retina. Given the crucial causative roles of oxidative stress and inflammation in AMD pathogenesis, knowing whether OTC might exhibit a similar benefit in this cell and tissue type has high clinical relevance; thus, we evaluated OTC in the present study. Methods ARPE-19 and primary RPE cells isolated from wild-type, Gpr109a−/−, or Slc5a8−/− mouse eyes were exposed to TNF-α in the presence or absence of OTC, followed by analysis of IL-6 and Ccl2 expression with real-time quantitative polymerase chain reaction or enzyme-linked immunosorbent assay. Cellular and molecular markers of inflammation and oxidative stress (i.e., IL-1β, TGF-β, ABCG1, ABCA1, reduced glutathione, and dihydroethidium) were evaluated in Ccl2−/−/Cx3cr1−/− double knockout mice on rd8 background (DKO rd8) treated with OTC (10 mg/ml) in drinking water for a period of 5 months. Results OTC treatment significantly inhibited the expression and secretion of IL-6 and Ccl2 in TNF-α-stimulated ARPE-19 cells. Studies conducted using DKO rd8 animals treated with OTC in drinking water confirmed these findings. Cellular and molecular markers of inflammation were significantly suppressed in the retinas of the OTC-treated DKO rd8 animals. Subsequent in vitro and in vivo studies of the possible mechanism(s) to explain these actions revealed that although OTC is an agonist of the anti-inflammatory G-protein coupled receptor GPR109A and a transportable substrate of the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), these properties may play a role but do not explain entirely the anti-inflammatory effects this compound elicits in cultured RPE cells and the intact mouse retina. Conclusions This study represents, to our knowledge, the first report of the suppressive effects of OTC on inflammation in cultured RPE cells and on inflammation and oxidative stress in the retina in vivo. PMID:24426777

Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart

PubMed Central

Maskell, Lauren J; Qamar, Kashif; Babakr, Aram A; Hawkins, Thomas A; Heads, Richard J; Budhram-Mahadeo, Vishwanie S

2017-01-01

Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a−/− : Brn-3b−/−) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts. PMID:28594399

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone*

PubMed Central

Schwarzer, Christian; Fu, Zhu; Morita, Takeshi; Whitt, Aaron G.; Neely, Aaron M.; Li, Chi; Machen, Terry E.

2015-01-01

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca2+ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca2+] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca2+ release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12. PMID:25627690

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

PubMed Central

Rudnick, Noam D.; Griffey, Christopher J.; Guarnieri, Paolo; Gerbino, Valeria; Wang, Xueyong; Piersaint, Jason A.; Tapia, Juan Carlos; Rich, Mark M.; Maniatis, Tom

2017-01-01

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression. PMID:28904095

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

PubMed Central

Pan, Yaoqian; Balazs, Louisa; Tigyi, Gabor; Yue, Junming

2013-01-01

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation. PMID:21371421

Pigment epithelium-derived factor reduces apoptosis and pro-inflammatory cytokine gene expression in a murine model of focal retinal degeneration.

PubMed

Wang, Yujuan; Subramanian, Preeti; Shen, Defen; Tuo, Jingsheng; Becerra, S Patricia; Chan, Chi-Chao

2013-11-26

AMD (age-related macular degeneration) is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. PEDF (pigment epithelium-derived factor) is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in DKO rd8 (Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)]) mice, a model for progressive, focal rd (retinal degeneration). First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and RPE (retinal pigment epithelium) than WT (wild-type, C57BL/6N). Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 μg), followed by a subconjunctival injection of PEDF (3 μg) 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E {[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium} levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. In addition, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both apoptotic and inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.

Development of Turbulence Models for Shear Flows by a Double Expansion technique.

DTIC Science & Technology

1991-07-01

Let us introduce the dimensionless variables 7 = -; I -_o E ~ Ko where S = (2Sij Sij)112 and K0 is the initial turbulent kinetic energy. In...simulation of Bardina eta 1 4 for co/ SKo = 0.296 Figure 2 19 IlI H- L -1 y x Turbulent flow over a backward facing step Figure 3 20 BACKWARD-FACING STEP

Methodology of Calculation the Terminal Settling Velocity Distribution of Spherical Particles for High Values of the Reynold's Number

NASA Astrophysics Data System (ADS)

Surowiak, Agnieszka; Brożek, Marian

2014-03-01

The particle settling velocity is the feature of separation in such processes as flowing classification and jigging. It characterizes material forwarded to the separation process and belongs to the so-called complex features because it is the function of particle density and size. i.e. the function of two simple features. The affiliation to a given subset is determined by the values of two properties and the distribution of such feature in a sample is the function of distributions of particle density and size. The knowledge about distribution of particle settling velocity in jigging process is as much important factor as knowledge about particle size distribution in screening or particle density distribution in dense media beneficiation. The paper will present a method of determining the distribution of settling velocity in the sample of spherical particles for the turbulent particle motion in which the settling velocity is expressed by the Newton formula. Because it depends on density and size of particle which are random variable of certain distributions, the settling velocity is a random variable. Applying theorems of probability, concerning distributions function of random variables, the authors present general formula of probability density function of settling velocity for the turbulent motion and particularly calculate probability density function for Weibull's forms of frequency functions of particle size and density. Distribution of settling velocity will calculate numerically and perform in graphical form. The paper presents the simulation of calculation of settling velocity distribution on the basis of real distributions of density and projective diameter of particles assuming that particles are spherical. Prędkość opadania ziarna jest cechą rozdziału w takich procesach przeróbki surowców jak klasyfikacja czy wzbogacanie w osadzarce. Cecha ta opisuje materiał kierowany do procesu rozdziału i należy do tzw. cech złożonych, ze względu na to, że jest funkcją dwóch cech prostych, którymi są: wielkość ziarna i gęstość ziarna. Przynależność do określonego podzbioru ziaren jest określona przez wartość dwóch cech, a rozkład tych cech w próbce jest funkcją rozkładów gęstości i wielkości ziarna. Znajomość rozkładu prędkości opadania ziaren w osadzarce jest istotnym parametrem jak znajomość rozkładu wielkości ziarna w procesie przesiewania czy znajomość rozkładu gęstości w procesie wzbogacania w cieczach ciężkich. W artykule przedstawiono metodykę wyliczania rozkładu prędkości opadania ziaren sferycznych w warunkach ruchu turbulentnego wyrażonego przy pomocy równania Newtona. Zarówno gęstość jak i wielkość ziarna są zmiennymi losowymi o określonych rozkładach. W związku z tym prędkość opadania ziarna jako funkcja cech prostych tj. gęstości i wielkości ziarna będzie również zmienną losową o rozkładzie, który jest funkcją rozkładów argumentów prostych. Wykorzystując twierdzenia rachunku prawdopodobieństwa odnoszące się do rozkładów funkcji zmiennych losowych przedstawiono ogólny wzór na funkcję gęstości rozkładu prędkości opadania w warunkach ruchu turbulentnego. Empiryczne rozkłady wielkości i gęstości ziaren aproksymowano rozkładem Weibulla. Rozkład prędkości opadania wyliczono numerycznie i przedstawiono w postaci graficznej. W artykule przedstawiono symulację wyliczania rozkładu prędkości opadania w oparciu o rzeczywiste rozkłady gęstości i średnicy projekcyjnej ziaren zakładając, że ziarna mają kształt sferyczny.

Muscle-specific AMPK β1β2-null mice display a myopathy due to loss of capillary density in nonpostural muscles

PubMed Central

Thomas, Melissa M.; Wang, David C.; D'Souza, Donna M.; Krause, Matthew P.; Layne, Andrew S.; Criswell, David S.; O'Neill, Hayley M.; Connor, Michael K.; Anderson, Judy E.; Kemp, Bruce E.; Steinberg, Gregory R.; Hawke, Thomas J.

2014-01-01

AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK β1β2 double-knockout (β1β2M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary β1β2M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, β1β2M-KO TA muscles displayed significant (P<0.05) increases in platelet aggregation and apoptosis within myofibers and surrounding interstitium (P<0.05). These changes correlated with a 45% decrease in capillary density (P<0.05). We hypothesized that the β1β2M-KO myopathy in resting muscle resulted from impaired AMPK-nNOSμ signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOSμ was decreased in β1β2M-KO compared to wild-type (WT) mice. The AMPK-nNOSμ relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) to β1β2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOSμ phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in β1β2M-KO muscles. These findings highlight the importance of the AMPK-nNOSμ pathway in resting skeletal muscle.—Thomas, M. M., Wang, D. C., D'Souza, D. M., Krause, M. P., Layne, A. S., Criswell, D. S., O'Neill, H. M., Connor, M. K., Anderson, J. E., Kemp, B. E., Steinberg, G. R., and Hawke, T. J. Muscle-specific AMPK β1β2-null mice display a myopathy due to loss of capillary density in nonpostural muscles. PMID:24522207

NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

PubMed

Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

2003-08-01

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

12 CFR 217.135 - Guarantees and credit derivatives: double default treatment.

Code of Federal Regulations, 2014 CFR

2014-01-01

...: KDD = Ko × (0.15 + 160 × PDg), Where: (1) ER11OC13.048 (2) PDg = PD of the protection provider. (3... formula for b provided in Table 1 in § 217.131, with PD equal to the lesser of PDo and PDg; and (7) M... mitigation benefits of a guarantee or credit derivative covering an exposure described in § 217.134(a)(1) by...

12 CFR 324.135 - Guarantees and credit derivatives: Double default treatment.

Code of Federal Regulations, 2014 CFR

2014-01-01

... to the following formula: KDD = Ko × (0.15 + 160 × PDg), where: (1) er10se13.039 (2) PDg equals PD of... appropriate formula for (R) provided in Table 1 in § 324.131, with PD equal to PDo. (6) b (maturity adjustment coefficient) is calculated according to the formula for b provided in Table 1 in § 324.131, with PD equal to...

Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion.

PubMed

Turner, Christopher J; Badu-Nkansah, Kwabena; Hynes, Richard O

2017-11-01

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply

PubMed Central

Krehl, Susanne; Loewinger, Maria; Florian, Simone; Kipp, Anna P.; Banning, Antje; Wessjohann, Ludger A.; Brauer, Martin N.; Iori, Renato; Esworthy, Robert S.; Chu, Fong-Fong; Brigelius-Flohé, Regina

2012-01-01

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective. PMID:22180572

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

PubMed

Krehl, Susanne; Loewinger, Maria; Florian, Simone; Kipp, Anna P; Banning, Antje; Wessjohann, Ludger A; Brauer, Martin N; Iori, Renato; Esworthy, Robert S; Chu, Fong-Fong; Brigelius-Flohé, Regina

2012-03-01

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response

PubMed Central

Gopalakrishnan, Anusha M.; Aly, Ahmed S. I.; Aravind, L.

2017-01-01

ABSTRACT In sexually reproducing organisms, meiosis is an essential step responsible for generation of haploid gametes from diploid somatic cells. The quest for understanding regulatory mechanisms of meiotic recombination in Plasmodium led to identification of a gene encoding a protein that contains 11 copies of C2H2 zinc fingers (ZnF). Reverse genetic approaches were used to create Plasmodium berghei parasites either lacking expression of full-length Plasmodium berghei zinc finger protein (PbZfp) (knockout [KO]) or expressing PbZfp lacking C-terminal zinc finger region (truncated [Trunc]). Mice infected with KO parasites survived two times longer (P < 0.0001) than mice infected with wild-type (WT) parasites. In mosquito transmission experiments, the infectivity of KO and Trunc parasites was severely compromised (>95% oocyst reduction). KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C2H2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination. PMID:28851851

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

PubMed

Li, Lijun; Byrd, Marcus; Doh, Kwame; Dixon, Patrice D; Lee, Hwal; Tiwari, Swasti; Ecelbarger, Carolyn M

2016-12-01

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated Akt T308 and IR Y 1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-β (TGF-β) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

KLF2 and KLF4 control endothelial identity and vascular integrity

PubMed Central

Sangwung, Panjamaporn; Zhou, Guangjin; Nayak, Lalitha; Chan, E. Ricky; Kang, Dong-Won; Zhang, Rongli; Lu, Yuan; Sugi, Keiki; Fujioka, Hisashi; Shi, Hong; Lapping, Stephanie D.; Ghosh, Chandra C.; Higgins, Sarah J.; Parikh, Samir M.; Jain, Mukesh K.

2017-01-01

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal. PMID:28239661

Further Development of Ko Displacement Theory for Deformed Shape Predictions of Nonuniform Aerospace Structures

NASA Technical Reports Server (NTRS)

Ko, William L.; Fleischer, Van Tran

2009-01-01

The Ko displacement theory previously formulated for deformed shape predictions of nonuniform beam structures is further developed mathematically. The further-developed displacement equations are expressed explicitly in terms of geometrical parameters of the beam and bending strains at equally spaced strain-sensing stations along the multiplexed fiber-optic sensor line installed on the bottom surface of the beam. The bending strain data can then be input into the displacement equations for calculations of local slopes, deflections, and cross-sectional twist angles for generating the overall deformed shapes of the nonuniform beam. The further-developed displacement theory can also be applied to the deformed shape predictions of nonuniform two-point supported beams, nonuniform panels, nonuniform aircraft wings and fuselages, and so forth. The high degree of accuracy of the further-developed displacement theory for nonuniform beams is validated by finite-element analysis of various nonuniform beam structures. Such structures include tapered tubular beams, depth-tapered unswept and swept wing boxes, width-tapered wing boxes, and double-tapered wing boxes, all under combined bending and torsional loads. The Ko displacement theory, combined with the fiber-optic strain-sensing system, provide a powerful tool for in-flight deformed shape monitoring of unmanned aerospace vehicles by ground-based pilots to maintain safe flights.

Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System.

PubMed

Nilaratanakul, Voraphoj; Chen, Jie; Tran, Oanh; Baxter, Victoria K; Troisi, Elizabeth M; Yeh, Jane X; Griffin, Diane E

2018-04-01

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID -/- ), secretory IgM-deficient (sIgM -/- ), and AID -/- sIgM -/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID -/- mice produce only germ line IgM, while sIgM -/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM -/- mice, but disease severity, weight loss, and survival were similar between the groups. AID -/- mice produced high levels of SINV-specific IgM, while sIgM -/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS. IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome. Copyright © 2018 American Society for Microbiology.

Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

PubMed

Susick, Laura L; Lowing, Jennifer L; Bosse, Kelly E; Hildebrandt, Clara C; Chrumka, Alexandria C; Conti, Alana C

2014-08-01

Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the maturational parameters examined in the current study may not be sensitive enough to detect effects of a single ethanol exposure during the brain growth spurt period. Genetic deletion of AC1/8 reveals a role for these cylases in attenuating ethanol-induced behavioral effects in the neonatally-exposed adolescent. Published by Elsevier B.V.

FAT10 knock out mice livers fail to develop Mallory-Denk bodies in the DDC mouse model.

PubMed

French, S W; French, B A; Oliva, J; Li, J; Bardag-Gorce, F; Tillman, B; Canaan, A

2012-12-01

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases β 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice. Copyright © 2012 Elsevier Inc. All rights reserved.

Estrogen via estrogen receptor beta partially inhibits mandibular condylar cartilage growth.

PubMed

Chen, J; Kamiya, Y; Polur, I; Xu, M; Choi, T; Kalajzic, Z; Drissi, H; Wadhwa, S

2014-11-01

Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression. Copyright © 2014 China University of Geosciences (Beijing) and Peking University. Published by Elsevier Ltd. All rights reserved.

Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response

PubMed Central

Marques, Joao T.; Rebouillat, Dominique; Ramana, Chilakamarti V.; Murakami, Junko; Hill, Jason E.; Gudkov, Andrei; Silverman, Robert H.; Stark, George R.; Williams, Bryan R. G.

2005-01-01

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication. PMID:16103161

Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.

PubMed

Fukuhara, Takasuke; Wada, Masami; Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-12-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

PubMed Central

Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-01-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly. PMID:25502789

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

PubMed

Ahmed, Emad A; Scherthan, Harry; de Rooij, Dirk G

2015-12-16

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

PubMed Central

Maddala, Rupalatha; Nagendran, Tharkika; Lang, Richard A.; Morozov, Alexei; Rao, Ponugoti V.

2015-01-01

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis. PMID:26212757

Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.

PubMed

Mao, Xiaoli; Wu, Yunyun; Diao, Huitian; Hao, Jianlei; Tian, Gaofei; Jia, Zhenghu; Li, Zheng; Xiong, Sidong; Wu, Zhenzhou; Wang, Puyue; Zhao, Liqing; Yin, Zhinan

2014-11-01

Our aim is to reveal the role of interleukin 6 (IL-6) in the pathogenesis of systemic lupus erythematosus (SLE) in a murine model of SLE. Normal female C57BL/6 mice were immunized with syngeneic-activated lymphocyte-derived DNA (ALD-DNA) to induce SLE. Non-immunized mice were used as control. SLE-associated markers, including anti-double-stranded DNA (anti-dsDNA) Abs, urine protein, and kidney histopathology, were assayed to ensure the induction of the disease. Compared with control mice, ALD-DNA immunized mice exhibited high levels of anti-dsDNA Abs, IL-6 expression in vivo and in vitro. We also found that IL-6 knockout (IL-6KO) mice were resistant to ALD-DNA-induced SLE. The activation of CD4(+) T cells in immunized IL-6KO mice was lower than in immunized wild-type (Wt) mice. Intracellular cytokine staining showed that Foxp3 expression in immunized IL-6KO mice was higher than in immunized Wt mice, which might be associated with the disease severity. We further discovered that ALD-DNA-stimulated dendritic cells supernatants could result in higher IL-6 and TNF-α expression and could suppress Foxp3 expression. In addition, blocking IL-6 could up-regulate Foxp3 expression. Therefore, our findings show that IL-6 promotes the progression of SLE via suppressing Treg differentiation.

BAD knockout provides metabolic seizure resistance in a genetic model of epilepsy with sudden unexplained death in epilepsy.

PubMed

Foley, Jeannine; Burnham, Veronica; Tedoldi, Meghan; Danial, Nika N; Yellen, Gary

2018-01-01

Metabolic alteration, either through the ketogenic diet (KD) or by genetic alteration of the BAD protein, can produce seizure protection in acute chemoconvulsant models of epilepsy. To assess the seizure-protective role of knocking out (KO) the Bad gene in a chronic epilepsy model, we used the Kcna1 -/- model of epilepsy, which displays progressively increased seizure severity and recapitulates the early death seen in sudden unexplained death in epilepsy (SUDEP). Beginning on postnatal day 24 (P24), we continuously video monitored Kcna1 -/- and Kcna1 -/- Bad -/- double knockout mice to assess survival and seizure severity. We found that Kcna1 -/- Bad -/- mice outlived Kcna1 -/- mice by approximately 2 weeks. Kcna1 -/- Bad -/- mice also spent significantly less time in seizure than Kcna1 -/- mice on P24 and the day of death, showing that BadKO provides seizure resistance in a genetic model of chronic epilepsy. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

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

PubMed

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

2016-08-01

Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system, and increasing evidence supports its role in inflammation, stress, and tissue injury, including injury to the lung and brain. We aimed to investigate the effects of TLR4 on neuroinflammation due to the lung-brain interaction in mechanically ventilated mice. Male wild-type (WT) C57BL/6 and TLR4 knockout (TLR4 KO) mice were divided into three groups: (1) control group (C): spontaneous breathing; (2) anesthesia group (A): spontaneous breathing under anesthesia; and (3) mechanical ventilation group (MV): 6h of MV under anesthesia. The behavioral responses of mice were tested with fear conditioning tests. The histological changes in the lung and brain were assessed using hematoxylin-eosin (HE) staining. The level of TLR4 mRNA in tissue was measured using reverse transcription-polymerase chain reaction (RT-PCR). The levels of inflammatory cytokines were measured with an enzyme-linked immunosorbent assay (ELISA). Microgliosis, astrocytosis, and the TLR4 immunoreactivity in the hippocampus were measured by double immunofluorescence. MV mice exhibited impaired cognition, and this impairment was less severe in TLR4 KO mice than in WT mice. In WT mice, MV increased TLR4 mRNA expression in the lung and brain. MV induced mild lung injury, which was prevented in TLR4 KO mice. MV mice exhibited increased levels of inflammatory cytokines, increased microglia and astrocyte activation. Microgliosis was alleviated in TLR4 KO mice. MV mice exhibited increased TLR4 immunoreactivity, which was expressed in microglia and astrocytes. These results demonstrate that TLR4 is involved in neuroinflammation due to the lung-brain interaction and that TLR4 KO ameliorates neuroinflammation due to lung-brain interaction after prolonged MV. In addition, Administration of a TLR4 antagonist (100μg/mice) to WT mice also significantly attenuated neuroinflammation of lung-brain interaction due to prolonged MV. TLR4 antagonism may be a new and novel approach for the treatment and management of neuroinflammation in long-term mechanically ventilated patients. Copyright © 2016 Elsevier Inc. All rights reserved.

An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo.

PubMed

Xu, Wenqin; Stadler, Cynthia K; Gorman, Kristen; Jensen, Nathaniel; Kim, David; Zheng, HaoQiang; Tang, Shaohua; Switzer, William M; Pye, Geoffrey W; Eiden, Maribeth V

2013-07-09

Leukemia and lymphoma account for more than 60% of deaths in captive koalas (Phascolarctos cinereus) in northeastern Australia. Although the endogenizing gammaretrovirus koala endogenous retrovirus (KoRV) was isolated from these koalas, KoRV has not been definitively associated with leukemogenesis. We performed KoRV screening in koalas from the San Diego Zoo, maintained for more than 45 y with very limited outbreeding, and the Los Angeles Zoo, maintained by continuously assimilating captive-born Australian koalas. San Diego Zoo koalas are currently free of malignant neoplasias and were infected with only endogenous KoRV, which we now term subtype "KoRV-A," whereas Los Angeles Zoo koalas with lymphomas/leukemias are infected in addition to KoRV-A by a unique KoRV we term subtype "KoRV-B." KoRV-B is most divergent in the envelope protein and uses a host receptor distinct from KoRV-A. KoRV-B also has duplicated enhancer regions in the LTR associated with increased pathology in gammaretroviruses. Whereas KoRV-A uses the sodium-dependent phosphate transporter 1 (PiT1) as a receptor, KoRV-B employs a different receptor, the thiamine transporter 1 (THTR1), to infect cells. KoRV-B is transmitted from dam to offspring through de novo infection, rather than via genetic inheritance like KoRV-A. Detection of KoRV-B in native Australian koalas should provide a history, and a mode for remediation, of leukemia/lymphoma currently endemic in this population.

Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors.

PubMed

Sutton, Gregory M; Trevaskis, James L; Hulver, Matthew W; McMillan, Ryan P; Markward, Nathan J; Babin, M Josephine; Meyer, Emily A; Butler, Andrew A

2006-05-01

Loss of brain melanocortin receptors (Mc3rKO and Mc4rKO) causes increased adiposity and exacerbates diet-induced obesity (DIO). Little is known about how Mc3r or Mc4r genotype, diet, and obesity affect insulin sensitivity. Insulin resistance, assessed by insulin and glucose tolerance tests, Ser(307) phosphorylation of insulin receptor substrate 1, and activation of protein kinase B, was examined in control and DIO wild-type (WT), Mc3rKO and Mc4rKO C57BL/6J mice. Mc4rKO mice were hyperphagic and had increased metabolic efficiency (weight gain per kilojoule consumed) relative to WT; both parameters increased further on high-fat diet. Obesity of Mc3rKO was more dependent on fat intake, involving increased metabolic efficiency. Fat mass of DIO Mc3rKO and Mc4rKO was similar, although Mc4rKO gained weight more rapidly. Mc4rKO develop hepatic insulin resistance and severe hepatic steatosis with obesity, independent of diet. DIO caused further deterioration of insulin action in Mc4rKO of either sex and, in male Mc3rKO, compared with controls, associated with increased fasting insulin, severe glucose intolerance, and reduced insulin signaling in muscle and adipose tissue. DIO female Mc3rKO exhibited very modest perturbations in glucose metabolism and insulin sensitivity. Consistent with previous data suggesting impaired fat oxidation, both Mc3rKO and Mc4rKO had reduced muscle oxidative metabolism, a risk factor for weight gain and insulin resistance. Energy expenditure was, however, increased in Mc4rKO compared with Mc3rKO and controls, perhaps due to hyperphagia and metabolic costs associated with rapid growth. In summary, DIO affects insulin sensitivity more severely in Mc4rKO compared with Mc3rKO, perhaps due to a more positive energy balance.

KOALA RETROVIRUS: A REVIEW.

PubMed

Kinney, Matthew E; Pye, Geoffrey W

2016-06-01

Koala retrovirus (KoRV) is a gammaretrovirus that has been identified in both captive and free-ranging koalas ( Phascolarctos cinereus ) with variable geographic distribution in Australia. KoRV is capable of both exogenous and endogenous transmission, which provides an interesting research platform for scientists to study active retrovirus endogenization into a host genome and offers veterinary scientists an opportunity to examine the clinical consequences of KoRV infection in koalas. Causation between KoRV and frequently recognized clinical conditions associated with immune suppression and neoplasia in koalas has not been definitively established, however research continues to evaluate a potential association. Three KoRV variants, KoRV-A, KoRV-B, and KoRV-J, have been the most thoroughly described and preliminary evidence suggests KoRV variability may be fundamental in host pathogenicity. In addition to reviewing what is currently known about KoRV, this article discusses treatment, management, and future research directions.

Extracellular Ca2+-induced force restoration in K+-depressed skeletal muscle of the mouse involves an elevation of [K+]i: implications for fatigue

PubMed Central

Leader, John P.; Loiselle, Denis S.; Higgins, Amanda; Lin, Wei; Renaud, Jean-Marc

2015-01-01

We examined whether a Ca2+-K+ interaction was a potential mechanism operating during fatigue with repeated tetani in isolated mouse muscles. Raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.3 to 10 mM in K+-depressed slow-twitch soleus and/or fast-twitch extensor digitorum longus muscles caused the following: 1) increase of intracellular K+ activity by 20–60 mM (raised intracellular K+ content, unchanged intracellular fluid volume), so that the K+-equilibrium potential increased by ∼10 mV and resting membrane potential repolarized by 5–10 mV; 2) large restoration of action potential amplitude (16–54 mV); 3) considerable recovery of excitable fibers (∼50% total); and 4) restoration of peak force with the peak tetanic force-extracellular K+ concentration ([K+]o) relationship shifting rightward toward higher [K+]o. Double-sigmoid curve-fitting to fatigue profiles (125 Hz for 500 ms, every second for 100 s) showed that prior exposure to raised [K+]o (7 mM) increased, whereas lowered [K+]o (2 mM) decreased, the rate and extent of force loss during the late phase of fatigue (second sigmoid) in soleus, hence implying a K+ dependence for late fatigue. Prior exposure to 10 mM [Ca2+]o slowed late fatigue in both muscle types, but was without effect on the extent of fatigue. These combined findings support our notion that a Ca2+-K+ interaction is plausible during severe fatigue in both muscle types. We speculate that a diminished transsarcolemmal K+ gradient and lowered [Ca2+]o contribute to late fatigue through reduced action potential amplitude and excitability. The raised [Ca2+]o-induced slowing of fatigue is likely to be mediated by a higher intracellular K+ activity, which prolongs the time before stimulation-induced K+ efflux depolarizes the sarcolemma sufficiently to interfere with action potentials. PMID:25571990

A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases

PubMed Central

Karakikes, Ioannis; Termglinchan, Vittavat; Cepeda, Diana A.; Lee, Jaecheol; Diecke, Sebastian; Hendel, Ayal; Itzhaki, Ilanit; Ameen, Mohamed; Shrestha, Rajani; Wu, Haodi; Ma, Ning; Shao, Ning-Yi; Seeger, Timon; Woo, Nicole; Wilson, Kitchener D.; Matsa, Elena; Porteus, Matthew H.; Sebastiano, Vittorio; Wu, Joseph C.

2017-01-01

Rationale Targeted genetic engineering using programmable nucleases such as transcription activator–like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. Objective The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. Methods and Results By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout (KO) eighty-eight human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene KO. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the general utility of the TALEN-mediated KO technique, six individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a dilated cardiomyopathy (DCM)-causing mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes (iPSC-CMs), we demonstrated that the KO strategy ameliorates the DCM phenotype in vitro. In addition, we modeled the Holt-Oram syndrome (HOS) in iPSC-CMs in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. Conclusion Collectively, our study illustrates the powerful combination of iPSCs and genome editing technology for understanding the biological function of genes and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research. PMID:28246128

Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

PubMed

Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

2018-05-18

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

PubMed Central

Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

2017-01-01

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

Exfoliative dermatitis

MedlinePlus

Bolognia JL, Schaffer JV, Duncan KO, Ko CJ. Erythroderma. In: Bolognia JL, Schaffer JV, Duncan KO, Ko CJ, eds. Dermatology Essentials. Philadelphia, PA: Elsevier; 2014:chap 8. Habif TP. Exanthems and ...

The Influence of Primary Microenvironment on Prostate Cancer Osteoblastic Bone Lesion Development

DTIC Science & Technology

2015-09-01

for inhibiting PCa bone lesion development: 3a. Basic fibroblast growth factor (bFGF) in PC3 bone metastasis: bFGF was identified by cytokine...II receptor (TβRII) knockout (Tgfbr2 KO) mouse models. Col1creERT/Tgfbr2 KO (Col/Tgfbr2 KO), which have TGF-β signaling specific KO in fibroblasts ... fibroblasts and osteoblasts in the bone by Colcre/Tgfbr2 KO, or in the myeloid lineage cells, including osteoclasts in the bone by LysMcre/Tgfbr2 KO

Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma.

PubMed

Day, Yuan-Ji; Huang, Liping; Ye, Hong; Li, Li; Linden, Joel; Okusa, Mark D

2006-03-01

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency

PubMed Central

Zhang, De-Mao; Cui, Di-Xin; Xu, Ruo-Shi; Zhou, Ya-Chuan; Zheng, Li-Wei; Liu, Peng; Zhou, Xue-Dong

2016-01-01

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption. PMID:27357320

Endothelial Estrogen Receptor-α Does Not Protect Against Vascular Stiffness Induced by Western Diet in Female Mice.

PubMed

Manrique, Camila; Lastra, Guido; Ramirez-Perez, Francisco I; Haertling, Dominic; DeMarco, Vincent G; Aroor, Annayya R; Jia, Guanghong; Chen, Dongqing; Barron, Brady J; Garro, Mona; Padilla, Jaume; Martinez-Lemus, Luis A; Sowers, James R

2016-04-01

Consumption of a diet high in fat and refined carbohydrates (Western diet [WD]) is associated with obesity and insulin resistance, both major risk factors for cardiovascular disease (CVD). In women, obesity and insulin resistance abrogate the protection against CVD likely afforded by estrogen signaling through estrogen receptor (ER)α. Indeed, WD in females results in increased vascular stiffness, which is independently associated with CVD. We tested the hypothesis that loss of ERα signaling in the endothelium exacerbates WD-induced vascular stiffening in female mice. We used a novel model of endothelial cell (EC)-specific ERα knockout (EC-ERαKO), obtained after sequential crossing of the ERα double floxed mice and VE-Cadherin Cre-recombinase mice. Ten-week-old females, EC-ERαKO and aged-matched genopairs were fed either a regular chow diet (control diet) or WD for 8 weeks. Vascular stiffness was measured in vivo by pulse wave velocity and ex vivo in aortic explants by atomic force microscopy. In addition, vascular reactivity was assessed in isolated aortic rings. Initial characterization of the model fed a control diet did not reveal changes in whole-body insulin sensitivity, aortic vasoreactivity, or vascular stiffness in the EC-ERαKO mice. Interestingly, ablation of ERα in ECs reduced WD-induced vascular stiffness and improved endothelial-dependent dilation. In the setting of a WD, endothelial ERα signaling contributes to vascular stiffening in females. The precise mechanisms underlying the detrimental effects of endothelial ERα in the setting of a WD remain to be elucidated.

Sucrose-conditioned flavor preferences in sweet ageusic T1r3 and Calhm1 knockout mice.

PubMed

Sclafani, Anthony; Marambaud, Philippe; Ackroff, Karen

2014-03-14

The present study compared the ability of sweet ageusic T1r3 knockout (KO) and Calhm1 KO mice to acquire preferences for a sucrose-paired flavor as well as for unflavored sucrose. The KO and wildtype (WT) mice were given 24-h one-bottle access to 8% sucrose containing one flavor CS+, e.g., grape) and to water containing a different flavor (CS-, e.g., cherry) over 4 training days. In subsequent two-bottle tests with the flavors in water only, the T1r3 KO and Calhm1 KO mice, like WT mice, preferred the CS+ to the CS-. After training with flavored solutions, both KO groups also preferred unflavored 8% sucrose to water although Calhm1 KO mice required more sugar experience to match the preference of the T1r3 KO mice. These findings demonstrate that Calhm1 KO mice, like T1r3 KO mice and WT mice, are sensitive to the post-oral preference conditioning actions of sucrose and can discriminate sugar from water. Yet, despite their acquired sucrose preferences, the Calhm1 KO and T1r3 KO mice consumed only half as much sugar per day as did WT mice. Thus, sweet taste signaling elements are not needed in the gut for sugar conditioning, but sweet taste signaling in the mouth is essential for the full expression of sugar appetite. Copyright © 2013 Elsevier Inc. All rights reserved.

Koala retrovirus genotyping analyses reveal a low prevalence of KoRV-A in Victorian koalas and an association with clinical disease.

PubMed

Legione, Alistair R; Patterson, Jade L S; Whiteley, Pam; Firestone, Simon M; Curnick, Megan; Bodley, Kate; Lynch, Michael; Gilkerson, James R; Sansom, Fiona M; Devlin, Joanne M

2017-02-01

Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas. Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status. The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and 'wet bottom' (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98). Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.

Functional Role of Serotonin in Insulin Secretion in a Diet-Induced Insulin-Resistant State

PubMed Central

Kim, Kyuho; Oh, Chang-Myung; Ohara-Imaizumi, Mica; Park, Sangkyu; Namkung, Jun; Yadav, Vijay K.; Tamarina, Natalia A.; Roe, Michael W.; Philipson, Louis H.; Karsenty, Gerard; Nagamatsu, Shinya

2015-01-01

The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic β-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases β-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in β-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of β-cell-specific Htr2b−/− (Htr2b βKO), Htr3a−/− (Htr3a knock-out [KO]), and β-cell-specific tryptophan hydroxylase 1 (Tph1)−/− (Tph1 βKO) mice on a high-fat diet (HFD). Htr2b βKO, Htr3a KO, and Tph1 βKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 βKO mice developed glucose intolerance, but Htr2b βKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 βKO mice, and 5-HT treatment improved insulin secretion from Tph1 βKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in β-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state. PMID:25426873

Roles of Toll-like receptors in allogeneic islet transplantation.

PubMed

Ro, Han; Hong, Juho; Kim, Beom Seok; Lee, Eun Won; Kim, Myung-Gyu; Han, Kyu Hyun; Yeom, Hye-Jung; Lee, Eun Mi; Jeong, Jong Cheol; Oh, Kook-Hwan; Ahn, Curie; Yang, Jaeseok

2012-11-27

Toll-like receptors (TLRs) are involved in the rejection of solid organ allografts. However, the roles of TLRs in islets are still controversial. We investigated the roles of TLRs in donor islets together with those in recipients in allogeneic islet transplantation. To assess the roles of TLRs in either donor islets or recipients, allogeneic islet transplantation was performed using myeloid differentiation factor 88 (MyD88)-knockout (KO), TLR4-KO, or Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-KO mice. Both polyriboinosinic polyribocytidylic acid and lipopolysaccharide (LPS) stimulation induced the mRNA expression of regulated and normal T cell expressed and secreted, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, interleukin-8, and inducible nitric oxide synthase in murine islets, whereas the induction was attenuated in TRIF-KO, interferon-β promoter stimulator-1-KO, and TLR4-KO mice. When islets from MyD88-KO, TLR4-KO, or TRIF-KO C57BL/6 mice were transplanted to BALB/c recipients, graft survival was not better than that of wild-type (WT) islets. However, the survival of the MyD88-KO islet allograft was significantly prolonged when combined with anti-CD40L. In parallel, LPS stimulation in donor islets interfered with anti-CD40L blockade-mediated long-term survival of islet allografts in TLR4-KO recipients. LPS stimulation increased the perigraft infiltration of both T cells and macrophages. Then again, when islets from WT BALB/c mice were transplanted to MyD88-KO, TRIF-KO, or WT C57BL/6 mice, there was no difference in graft survival, although some of the MyD88-KO recipients obtained long-term graft survival. However, anti-CD40L prolonged graft survival significantly in MyD88-KO recipients. The absence of MyD88 in either donors or recipients decreased the perigraft infiltration of inflammatory cells when combined with anti-CD40L. TLRs in both donor islets and recipients are involved in islet allograft rejection.

Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models.

PubMed

Stantzou, Amalia; Ueberschlag-Pitiot, Vanessa; Thomasson, Remi; Furling, Denis; Bonnieu, Anne; Amthor, Helge; Ferry, Arnaud

2017-02-01

The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KO Lee ) and (ii) the Grobet model (KO Grobet ), and measured the contraction of tibialis anterior muscle in situ. Absolute maximal isometric force was increased in 6-month-old KO Lee and KO Grobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KO Lee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KO Grobet mice, whereas specific maximal power was reduced only in male KO Lee mice. Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017. © 2016 Wiley Periodicals, Inc.

Autonomic nervous system involvement in the giant axonal neuropathy (GAN) KO mouse: implications for human disease.

PubMed

Armao, Diane; Bailey, Rachel M; Bouldin, Thomas W; Kim, Yongbaek; Gray, Steven J

2016-08-01

Giant axonal neuropathy (GAN) is an inherited severe sensorimotor neuropathy. The aim of this research was to investigate the neuropathologic features and clinical autonomic nervous system (ANS) phenotype in two GAN knockout (KO) mouse models. Little is known about ANS involvement in GAN in humans, but autonomic signs and symptoms are commonly reported in early childhood. Routine histology and immunohistochemistry was performed on GAN KO mouse specimens taken at various ages. Enteric dysfunction was assessed by quantifying the frequency, weight, and water content of defecation in GAN KO mice. Histological examination of the enteric, parasympathetic and sympathetic ANS of GAN KO mice revealed pronounced and widespread neuronal perikaryal intermediate filament inclusions. These neuronal inclusions served as an easily identifiable, early marker of GAN in young GAN KO mice. Functional studies identified an age-dependent alteration in fecal weight and defecation frequency in GAN KO mice. For the first time in the GAN KO mouse model, we described the early, pronounced and widespread neuropathologic features involving the ANS. In addition, we provided evidence for a clinical autonomic phenotype in GAN KO mice, reflected in abnormal gastrointestinal function. These findings in GAN KO mice suggest that consideration should be given to ANS involvement in human GAN, especially when considering treatments and patient care.

Distinct function of estrogen receptor α in smooth muscle and fibroblast cells in prostate development.

PubMed

Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

2013-01-01

Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal and epithelial cells. Smooth muscle ERαKO (smERαKO) mice showed decreased glandular infolding with the proximal area exhibiting a significant decrease. Fibroblast ERαKO mouse prostates did not exhibit this phenotype but showed a decrease in the number of ductal tips. Additionally, the amount of collagen observed in the basement membrane was reduced in smERαKO prostates. Interestingly, these phenotypes were found to be mutually exclusive among smERαKO or fibroblast ERαKO mice. Compound KO of ERα in both fibroblast and smooth muscle showed combined phenotypes from each of the single KO. Further mechanistic studies showed that IGF-I and epidermal growth factor were down-regulated in prostate smooth muscle PS-1 cells lacking ERα. Together, our results indicate the distinct functions of fibroblast vs. smERα in prostate development.

Controllable Electrochemical Fabrication of KO2-Decorated Binder-Free Cathodes for Rechargeable Lithium-Oxygen Batteries.

PubMed

Yu, Wei; Wang, Huwei; Qin, Lei; Hu, Junyang; Liu, Liang; Li, Baohua; Zhai, Dengyun; Kang, Feiyu

2018-05-23

Understanding the electrochemical property of superoxides in alkali metal oxygen batteries is critical for the design of a stable oxygen battery with high capacity and long cycle performance. In this work, a KO 2 -decorated binder-free cathode is fabricated by a simple and efficient electrochemical strategy. KO 2 nanoparticles are uniformly coated on the carbon nanotube film (CNT-f) through a controllable discharge process in the K-O 2 battery, and the KO 2 -decorated CNT-f is innovatively introduced into the Li-O 2 battery as the O 2 diffusion electrode. The Li-O 2 battery based on the KO 2 -decorated CNT-f cathode can deliver enhanced discharge capacity, reduced charge overpotential, and more stable cycle performance compared with the battery in the absence of KO 2 . In situ formed KO 2 particles on the surface of CNT-f cathode assist to form Li 2 O 2 nanosheets in the Li-O 2 battery, which contributes to the improvement of discharge capacity and cycle life. Interestingly, the analysis of KO 2 -decorated CNT-f cathodes, after discharge and cycle tests, reveals that the electrochemically synthesized KO 2 seems not a conventional electrocatalyst but a partially dissolvable and decomposable promoter in Li-O 2 batteries.

Complete genome analysis of porcine kobuviruses from the feces of pigs in Japan.

PubMed

Akagami, Masataka; Ito, Mika; Niira, Kazutaka; Kuroda, Moegi; Masuda, Tsuneyuki; Haga, Kei; Tsuchiaka, Shinobu; Naoi, Yuki; Kishimoto, Mai; Sano, Kaori; Omatsu, Tsutomu; Aoki, Hiroshi; Katayama, Yukie; Oba, Mami; Oka, Tomoichiro; Ichimaru, Toru; Yamasato, Hiroshi; Ouchi, Yoshinao; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya; Nagai, Makoto

2017-08-01

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes.

PubMed

Turner, Peter C; Yomano, Lorraine P; Jarboe, Laura R; York, Sean W; Baggett, Christy L; Moritz, Brélan E; Zentz, Emily B; Shanmugam, K T; Ingram, Lonnie O

2012-04-01

Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

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

PubMed Central

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

2015-01-01

After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side-effects. PMID:26082754

Entomological evaluation of PermaNet 2.0® and K-O Tab 1-2-3® treated nets in comparison to nets conventionally treated with deltamethrin, after repeated washing.

PubMed

Kayedi, Mohammad Hassan; Khamisabadi, Kiumars; Dehghani, Nader; Haghdoost, Ali Akbar

2015-06-01

The residual insecticidal power of two types of ITNs (PermaNet 2.0® (PN2) and K-O Tab 1-2-3® (KO 123)), compared to K-O Tab® (KO) treated nets, was assessed. The nets were tested unwashed, and after being washed, by hand 5, 15 and 21 times, respectively. After each wash, the nets were dried vertically on a line, in the shade. Two types of bioassays (mean median knock down times (MMKDT) and mortality 24 hours after a 3-minute exposure (%mortality)) were used, along with reared female Anopheles stephensi. The number of washes had a great impact on MMKDT and %mortality of all types of nets. This impact was greater for conventionally treated nets, indicating that PN2 and KO 123 nets are significantly more wash resistant than KO nets after 21 washes. There was no significant difference between PN2 and KO 123 with respect to %mortality 24 hours after a 3-minute exposure at 0, 15 and 21 washes. Similarly, the same results were obtained for MMKDT, and the differences between PN2 and KO 123 were not statistically significant. This study demonstrates that the efficacy of KO 123 nets is as beneficial as the efficacy of PN2 nets up to 21 washes.

Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

2006-07-01

Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.

PubMed

Cuchel, Marina; Lund-Katz, Sissel; de la Llera-Moya, Margarita; Millar, John S; Chang, David; Fuki, Ilia; Rothblat, George H; Phillips, Michael C; Rader, Daniel J

2010-03-01

Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro. Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001). The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes.

PubMed

Okiyama, Naoko; Katz, Stephen I

2014-09-01

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases. Published by Elsevier Ltd.

Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

PubMed

Suzuki, Maiko; Ikeda, Atsushi; Bartlett, John D

2018-03-01

Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8 Sirt/KO ) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8 Sirt1/over ) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8 Sirt/KO cells, whereas it decreased in LS8 Sirt1/over . Fluoride-induced Ac-p53 formation was also suppressed in LS8 Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8 Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8 Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8 Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8 Sirt1/KO cells, but was attenuated in LS8 Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

Effect of the Continuous Traverse Trajectory and Dynamic Error of the Vane Anemometer on the Accuracy of Average Velocity Measurements at the Cross-Section of the Mine Heading - Model-Based Testing

NASA Astrophysics Data System (ADS)

Jamróz, Paweł

2014-12-01

This paper discusses the problem of measuring the average velocity at the cross-section of mine heading with the use of the continuous traverse method. Based on model testing, it has been shown that measurement signals, obtained along the traversing trajectory, belong to the group of non-stationary signals. The methodology of the traversing method measurements, with the aspect of capabilities of measuring instruments used for that purpose, has been analysed. Results of simulation tests concerning the dynamic response of a vane anemometer to the measurement signal for selected trajectories of the continuous traverse have been presented. For this purpose, a velocity profile presenting an expanded stream of undisturbed air flow in the excavation has been used. Attention has been paid to the problem of selecting an adequate trajectory of anemometer movement, as the value of the velocity measured at the cross-section depends on the trajectory W artykule poruszono problem pomiarów prędkości średniej w przekroju wyrobiska kopalnianego z wykorzystaniem metody trawersu ciągłego. Na podstawie badań modelowych wykazano, że sygnały pomiarowe, które uzyskuje się wzdłuż trajektorii trawersowania należą do grupy sygnałów niestacjonarnych. Przeanalizowano metodologię przeprowadzania pomiarów metodą trawersowania w aspekcie możliwości pomiarowych wykorzystywanych w tym celu urządzeń pomiarowych. Przedstawiono wyniki badań symulacyjnych dynamicznej odpowiedzi anemometru skrzydełkowego na sygnał pomiarowy dla wybranych trajektorii trawersu ciągłego. W tym celu wykorzystano profil prędkości przedstawiający rozwiniętą strugę niezakłóconego przepływu powietrza w chodniku. Zwrócono uwagę na problem doboru odpowiedniej trajektorii przemieszczania anemometru, od której zależy wartość mierzonej prędkości średniej w przekroju.

Type II iodothyronine deiodinase provides intracellular 3,5,3'-triiodothyronine to normal and regenerating mouse skeletal muscle.

PubMed

Marsili, Alessandro; Tang, Dan; Harney, John W; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico; Larsen, P Reed

2011-11-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-01-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

SMAD Signaling Is Required for Structural Integrity of the Female Reproductive Tract and Uterine Function During Early Pregnancy in Mice1

PubMed Central

Rodriguez, Amanda; Tripurani, Swamy K.; Burton, Jason C.; Clementi, Caterina; Larina, Irina; Pangas, Stephanie A.

2016-01-01

Pregnancy is a complex physiological process tightly controlled by the interplay among hormones, morphogens, transcription factors, and signaling pathways. Although recent studies using genetically engineered mouse models have revealed that ligands and receptors of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) signaling pathways are essential for multiple reproductive events during pregnancy, the functional role of SMAD transcription factors, which serve as the canonical signaling platform for the TGFbeta/BMP pathways, in the oviduct and uterus is undefined. Here, we used a mouse model containing triple conditional deletion of the BMP receptor signaling Smads (Smad1 and Smad5) and Smad4, the central mediator of both TGFbeta and BMP signaling, to investigate the role of the SMADs in reproductive tract structure and function in cells from the Amhr2 lineage. Unlike the respective single- or double-knockouts, female Smad1flox/flox Smad5flox/flox Smad4flox/flox Amhr2cre/+conditional knockout (i.e., Smad1/5/4-Amhr2-cre KO) mice are sterile. We discovered that Smad1/5/4-Amhr2-cre KO females have malformed oviducts that subsequently develop oviductal diverticuli. These oviducts showed dysregulation of multiple genes essential for oviduct and smooth muscle development. In addition, uteri from Smad1/5/4-Amhr2-cre KO females exhibit multiple defects in stroma, epithelium, and smooth muscle layers and fail to assemble a closed uterine lumen upon embryo implantation, with defective uterine decidualization that led to pregnancy loss at early to mid-gestation. Taken together, our study uncovers a new role for the SMAD transcription factors in maintaining the structural and functional integrity of oviduct and uterus, required for establishment and maintenance of pregnancy. PMID:27335065

Type II iodothyronine deiodinase provides intracellular 3,5,3′-triiodothyronine to normal and regenerating mouse skeletal muscle

PubMed Central

Marsili, Alessandro; Tang, Dan; Harney, John W.; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico

2011-01-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T4) to 3,5,3′-triiodothyronine (T3), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T3 under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T4-to-T3 conversion increases during differentiation in C2C12 myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given 125I-T4 and 131I-T3, the intracellular 125I-T3/131I-T3 ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the 125I-T3/131I-T3 ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in 125I-T3/131I-T3 ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of 125I-T3 is doubled after skeletal muscle injury. Thus, D2-mediated T4-to-T3 conversion generates significant intracellular T3 in normal mouse skeletal muscle, with the increased T3 required for muscle regeneration being provided by increased D2 synthesis, not by T3 from the circulation. PMID:21771965

Increased vascular sensitivity and connexin43 expression after sympathetic denervation.

PubMed

Slovut, David P; Mehta, Shyamal H; Dorrance, Anne M; Brosius, Frank C; Watts, Stephanie W; Webb, R Clinton

2004-05-01

Following denervation, arteries demonstrate a heightened sensitivity to alpha-adrenergic agonists and increased oscillatory contractions that may partly result from increased gap junction expression. Hence, we wanted to study the effect of sympathetic denervation on connexin43 (Cx43) expression and agonist-induced contractility in the vascular smooth muscle (VSM). Effects of denervation with reserpine (3 mg/kg/day, i.p.) or topical 5% phenol-glycerol on VSM contractions and expression of the gap junction Cx43 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting for Cx43 protein were examined. Wistar-Kyoto (WKY) rat tail arteries were exposed to norepinephrine (NE) (10(-9)-10(-5) M). Reactivity was also examined in the carotid arteries and thoracic aortas from Cx43 heterozygote deficient (KO) mice. The concentration for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (p<0.05). NE-induced oscillatory activity (OA) was seen in 5/5 reserpine- and 5/8 phenol-treated vessels vs. 0/12 controls (p<0.05). Spontaneous OA was observed more frequently in carotid and aortic rings from WT than Cx43 KO rings. Cumulative OA in response to alpha-adrenergic stimulation was significantly greater in WT carotid (429+/-101 vs. 128+/-7 mN s, p<0.05) and aortic rings (337+/-85 vs. 134+/-11 mN s, p<0.05) than in Cx43 KO rings. Following denervation, RT-PCR showed significantly increased levels of Cx43 mRNA (p<0.05). Western blot analysis revealed near doubling of Cx43 protein (p<0.05). We conclude that sympathetic denervation results in increased expression of Cx43, which in turn, contributes to increased spontaneous and agonist-induced OA in VSM.

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Yaoqian; Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163; Balazs, Louisa

2011-05-13

Highlights: {yields} Deletion of Dicer in vascular smooth muscle cells(VSMCs) leads to embryonic mortality. {yields} Loss of Dicer in VSMCs leads to developmental delay. {yields} Loss of Dicer in VSMCs leads to hemorrhage in various organs including brain, skin and liver. {yields} Loss of Dicer in VSMCs leads to vascular wall remodeling. {yields} Loss of Dicer in VSMCs dysregulates the expression of miRNA and VSMC marker genes. -- Abstract: Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role ofmore » Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.« less

Nox2 Knockout Delays Infarct Progression and Increases Vascular Recovery through Angiogenesis in Mice following Ischaemic Stroke with Reperfusion

PubMed Central

McCann, Sarah K.; Dusting, Gregory J.; Roulston, Carli L.

2014-01-01

Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation. PMID:25375101

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

PubMed

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

2015-03-01

Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V̇(E)) and rate of O2 consumption ( V̇(O2)) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6 mg kg(-1) day(-1) beginning on embryonic day 14). Mice were exposed to seven 2 min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2 min of air. Compared to WT, α4 KO mice had increased air V̇(E) and V̇(O2) at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V̇(E)/V̇(O2)) to MA. At P1-2, nicotine suppressed air V̇(E) and V̇(O2) in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V̇(E) and V̇(O2) of only α4 KO's but also significantly enhanced V̇(E) during MA (nearly double that of WT; p<0.001). This study has revealed complex effects of α4 nAChR deficiency and prenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress. Copyright © 2015 Elsevier B.V. All rights reserved.

The Probiotic Lactobacillus Prevents Citrobacter rodentium-Induced Murine Colitis in a TLR2-Dependent Manner.

PubMed

Ryu, Seung-Hyun; Park, Jong-Hyung; Choi, Soo-Young; Jeon, Hee-Yeon; Park, Jin-Il; Kim, Jun-Young; Ham, Seung-Hoon; Choi, Yang-Kyu

2016-07-28

The main objective of this study was to investigate whether Lactobacillus rhamnosus GG (LGG) ameliorated the effects of Citrobactor rodentium infection in Toll-like receptor 2 (TLR2) knockout (KO) and TLR4 KO mice, as well as in wild-type C57BL/6 (B6) mice. TLR2 KO, TLR4 KO, and B6 mice were divided into three groups per each strain. Each group had an uninfected control group (n = 5), C. rodentium-infected group (n = 8), and LGG-pretreated C. rodentium-infected group (n = 8). The survival rate of B6 mice infected with C. rodentium was higher when pretreated with LGG. Pretreatment with LGG ameliorated C. rodentium-induced mucosal hyperplasia in B6 and TLR4 KO mice. However, in C-rodentium-infected TLR2 KO mice, mucosal hyperplasia persisted, regardless of pretreatment with LGG. In addition, LGG-pretreated B6 and TLR4 KO mice showed a decrease in spleen weight and downregulation of tumor necrosis factor alpha, interferon gamma, and monocyte chemotactic protein 1 mRNA expression compared with the non-pretreated group. In contrast, such changes were not observed in TLR2 KO mice, regardless of pretreatment with LGG. From the above results, we conclude that pretreatment with LGG ameliorates C. rodentium-induced colitis in B6 and TLR4 KO mice, but not in TLR2 KO mice. Therefore, LGG protects mice from C. rodentium-induced colitis in a TLR2-dependent manner.

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.

PubMed

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

2017-11-01

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

Trps1 deficiency inhibits the morphogenesis of secondary hair follicles via decreased Noggin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yujing; Nakanishi, Masako; Sato, Fuyuki

Highlights: • The number of secondary hair follicles is reduced by half in Trps1 KO embryonic skin compared to wild-type skin. • Noggin expression is significantly decreased and BMP signaling is promoted in Trps1 KO embryonic skin. • Treatment with a Noggin or BMP inhibitor rescued the decreased number of hair follicles in Trps1 KO skin graft cultures. • Cell proliferation and apoptosis of the epidermis were normalized by Noggin treatment. - Abstract: A representative phenotype of patients with tricho-rhino-phalangeal syndrome (TRPS) is sparse hair. To understand the developmental defects of these patient’s hair follicles, we analyzed the development ofmore » hair follicles histologically and biochemically using Trps1 deficient (KO) mice. First, we compared the numbers of primary hair follicles in wild-type (WT) and KO embryos at different developmental stages. No differences were observed in the E14.5 skins of WT and KO mice. However, at later time points, KO fetal skin failed to properly develop secondary hair follicles, and the number of secondary hair follicles present in E18.5 KO skin was approximately half compared to that of WT skin. Sonic hedgehog expression was significantly decreased in E17.5 KO skin, whereas no changes were observed in Eda/Edar expression in E14.5 or E17.5 skins. In addition, Noggin expression was significantly decreased in E14.5 and E17.5 KO skin compared to WT skin. In parallel with the suppression of Noggin expression, BMP signaling was promoted in the epidermal cells of KO skins compared to WT skins as determined by immunohistochemistry for phosphorylated Smad1/5/8. The reduced number of secondary hair follicles was restored in skin graft cultures treated with a Noggin and BMP inhibitor. Furthermore, decreased cell proliferation, and increased apoptosis in KO skin was rescued by Noggin treatment. Taken together, we conclude that hair follicle development in Trps1 KO embryos is impaired directly or indirectly by decreased Noggin expression.« less

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

PubMed

Chappell, K J; Brealey, J C; Amarilla, A A; Watterson, D; Hulse, L; Palmieri, C; Johnston, S D; Holmes, E C; Meers, J; Young, P R

2017-02-01

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance. Copyright © 2017 American Society for Microbiology.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrill, Joshua A.; Hukkanen, Renee R.; Lawson, Marie

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 expressionmore » 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 development are observed in AHR-KO mouse, but not rat. • Renal pathology is observed in AHR-KO rat, but not mouse.« less

Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

PubMed

Luhrs, Lauren; Manlapaz, Cynthia; Kedzie, Karen; Rao, Sandhya; Cabrera-Ghayouri, Sara; Donello, John; Gil, Daniel

2016-12-17

Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and obsessive compulsive disorder. Copyright © 2016. Published by Elsevier Ltd.

Differentiation of Forebrain and Hippocampal Dopamine 1-Class Receptors, D1R and D5R, in Spatial Learning and Memory

PubMed Central

Sariñana, Joshua; Tonegawa, Susumu

2017-01-01

Activation of prefrontal cortical (PFC), striatal, and hippocampal dopamine 1-class receptors (D1R and D5R) is necessary for normal spatial information processing. Yet the precise role of the D1R versus the D5R in the aforementioned structures, and their specific contribution to the water-maze spatial learning task remains unknown. D1R- and D5R- specific in situ hybridization probes showed that forebrain restricted D1R and D5R KO mice (F-D1R/D5R KO) displayed D1R mRNA deletion in the medial (m)PFC, dorsal and ventral striatum, and the dentate gyrus (DG) of the hippocampus. D5R mRNA deletion was limited to the mPFC, the CA1 and DG hippocampal subregions. F-D1R/D5R KO mice were given water-maze training and displayed subtle spatial latency differences between genotypes and spatial memory deficits during both regular and reversal training. To differentiate forebrain D1R from D5R activation, forebrain restricted D1R KO (F-D1R KO) and D5R KO (F-D5R KO) mice were trained on the water-maze task. F-D1R KO animals exhibited escape latency deficits throughout regular and reversal training as well as spatial memory deficits during reversal training. F-D1R KO mice also showed perseverative behavior during the reversal spatial memory probe test. In contrast, F-D5R KO animals did not present observable deficits on the water-maze task. Because F-D1R KO mice showed water-maze deficits we tested the necessity of hippocampal D1R activation for spatial learning and memory. We trained DG restricted D1R KO (DG-D1R KO) mice on the water-maze task. DG-D1R KO mice did not present detectable spatial memory deficit, but did show subtle deficits during specific days of training. Our data provides evidence that forebrain D5R activation plays a unique role in spatial learning and memory in conjunction with D1R activation. Moreover, these data suggest that mPFC and striatal, but not DG D1R activation are essential for spatial learning and memory. PMID:26174222

Extracellular K+ in the supraoptic nucleus of the rat during reflex bursting activity by oxytocin neurones.

PubMed Central

Coles, J A; Poulain, D A

1991-01-01

1. We have investigated changes in extracellular potassium concentration [K+]o in the supraoptic nucleus of lactating rats and in particular those that occur during the intense burst of firing by the oxytocin neurones involved in the milk ejection reflex. 2. Double-barrelled K(+)-selective microelectrodes containing a highly selective sensor based on valinomycin were lowered through the exposed cortex towards the supraoptic nucleus (SON) of female rats anaesthetized with urethane. The mean resting [K+]o in the hypothalami of five rats was 2.4 mM, S.D. = 0.3 mM. 3. Where the reference barrel recorded extracellular action potentials from an oxytocin cell, the reflex burst of firing (4 s, typical maximum 50 Hz) was accompanied by a mean increase in [K+]o (delta[K+]o) of 0.22 mM (S.E.M. = 0.02 mM, fifty-seven bursts in eight cells in seven rats). The rise in [K+]o did not begin more than 0.1 s before the onset of the burst, and began to fall from its maximum during the burst. Slow field potentials, indicative of spatial buffering of K+, were undetectable (less than 50 microV). When the electrode was advanced in steps, the amplitudes of both delta[K+]o and the action potential declined steeply to about 10% over a distance of 20 microns: K+ from oxytocin cells appears to be prevented from dispersing freely through the extracellular space of the SON. 4. When the electrode recorded action potentials from a vasopressin cell, delta[K+]o during an oxytocin cell burst was very small: 0.021 mM (S.E.M. = 0.005 mM). At other sites in the SON, where antidromic stimulation evoked a field potential but no action potential, delta[K+]o was 0.047 +/- 0.005 mM. We conclude that the reason oxytocin bursts do not affect vasopressin cells is that [K+]o rises very little around vasopressin cells. A fortiori, since the increases in [K+]o were very small except where action potentials from oxytocin cells were recorded, they can make no significant contribution to synchronizing the onsets of bursts in oxytocin cells that are not contiguous. 5. A standard antidromic stimulation from the pituitary stalk, at 40 Hz for 4 s, which stimulated both oxytocin neurones and vasopressin neurones, caused a delta[K+]o of 0.17-1.8 mM, the variation being mainly from rat to rat. The larger delta[K+]o values were accompanied by slow negative potentials of up to 1.5 mV, there was a gradient in delta[K+]o decreasing towards the pia at the inferior limit of the SON, and there was a slow increase in [K+] in the subarachnoid space.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1895242

Detection of koala retrovirus subgroup B (KoRV-B) in animals housed at European zoos.

PubMed

Fiebig, Uwe; Keller, Martina; Denner, Joachim

2016-12-01

Many koalas carry an endogenous retrovirus, KoRV-A, in their genome. Recently, a second retrovirus, KoRV-B, was detected in koalas in Japanese and U.S. zoos. However, this virus is not endogenous, differs in the receptor binding site of the surface envelope protein, and uses a receptor different from that of KoRV-A. We describe here a KoRV-B found in koalas at zoos in Germany and Belgium that differs slightly from that found in the Los Angeles zoo.

Inactivation of the maternal fragile X gene results in sensitization of GABAB receptor function in the offspring.

PubMed

Zupan, Bojana; Toth, Miklos

2008-12-01

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR1 gene, with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Although fragile X syndrome is considered a typical Mendelian disorder, we have recently reported that the environment, specifically the fmr1(+/-) or fmr1(-/-) [H or knockout (KO)] maternal environment, elicits on its own a partial fragile X-like phenotype and can contribute to the overall phenotype of fmr1(-/0) (KO) male offspring. Genetically fmr1(+/0) (WT) males born to H females (H(maternal) > WT(offspring)), similar to KO male offspring born to H and KO mothers (H > KO and KO > KO), exhibit locomotor hyperactivity. These mice also showed reduced D(2) autoreceptor function, indicating a possible diminished feedback inhibition of dopamine (DA) release in the nigrostriatal and mesolimbic systems. The GABAergic system also regulates DA release, in part via presynaptic GABA(B) receptors (Rs) located on midbrain dopaminergic neurons. Here, we show that the locomotor inhibitory effect of the GABA(B)R agonist baclofen [4-amino-3-(4-chlorophenyl)-butanoic acid] is enhanced in all progeny of mutant mothers (H > WT, H > KO, and KO > KO) compared with WT > WT mice, irrespective of their own genotype. However, increased sensitivity to baclofen was selective and limited to the locomotor response because the muscle-relaxant and sedative effects of the drug were not altered by the maternal environment. These data show that GABA(B)R sensitization, traditionally induced pharmacologically, can also be elicited by the fmr1-deficient maternal environment.

An Essential Physiological Role for MCT8 in Bone in Male Mice

PubMed Central

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.

2017-01-01

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. PMID:28637283

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

PubMed

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

2015-11-01

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

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

PubMed

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.

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis

PubMed Central

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P.; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-01-01

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis. PMID:28191821

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-02-13

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Wild-type male offspring of fmr-1+/- mothers exhibit characteristics of the fragile X phenotype.

PubMed

Zupan, Bojana; Toth, Miklos

2008-10-01

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR-1 gene with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Males are more severely affected than heterozygote (H) females, who, as carriers, have a 50% chance of transmitting the mutated allele in each pregnancy. fmr-1 knockout (KO) mice reproduce fragile X symptoms, including hyperactivity, seizures, and abnormal sensory processing. In contrast to the expectation that wild-type (WT) males born to H (fmr-1(+/-)) mothers (H>WT) are behaviorally normal and indistinguishable from WT males born to WT mothers (WT>WT); here, we show that H>WT offspring are more active than WT>WT offspring and that their hyperactivity is similar to male KO mice born to H or KO (fmr-1(-/-)) mothers (H>KO/KO>KO). H>WT mice, however, do not exhibit seizures or abnormal sensory processing. Consistent with their hyperactivity, the effect of the D2 agonist quinpirole is reduced in H>WT as well as in H>KO and KO>KO mice compared to WT>WT offspring, suggesting a diminished feedback inhibition of dopamine release. Our data indicate that some aspects of hyperactivity and associated dopaminergic changes in 'fragile X' mice are a maternal fmr-1 genotype rather than an offspring fmr-1 genotype effect.

Wild-Type Male Offspring of fmr-1+/− Mothers Exhibit Characteristics of the Fragile X Phenotype

PubMed Central

Zupan, Bojana; Toth, Miklos

2009-01-01

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR-1 gene with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Males are more severely affected than heterozygote (H) females, who, as carriers, have a 50% chance of transmitting the mutated allele in each pregnancy. fmr-1 knockout (KO) mice reproduce fragile X symptoms, including hyperactivity, seizures, and abnormal sensory processing. In contrast to the expectation that wild-type (WT) males born to H (fmr-1+/−) mothers (H> WT) are behaviorally normal and indistinguishable from WT males born to WT mothers (WT> WT); here, we show that H> WT offspring are more active than WT> WT offspring and that their hyperactivity is similar to male KO mice born to H or KO (fmr-1−/−) mothers (H> KO/KO> KO). H> WT mice, however, do not exhibit seizures or abnormal sensory processing. Consistent with their hyperactivity, the effect of the D2 agonist quinpirole is reduced in H> WT as well as in H> KO and KO> KO mice compared to WT> WT offspring, suggesting a diminished feedback inhibition of dopamine release. Our data indicate that some aspects of hyperactivity and associated dopaminergic changes in ‘fragile X’ mice are a maternal fmr-1 genotype rather than an offspring fmr-1 genotype effect. PMID:18172434

Exercise ameliorates endoplasmic reticulum stress-mediated vascular dysfunction in mesenteric arteries in atherosclerosis.

PubMed

Hong, Junyoung; Kim, Kwangchan; Park, Eunkyung; Lee, Jonghae; Markofski, Melissa M; Marrelli, Sean P; Park, Yoonjung

2018-05-21

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

Effects of long-term intake of Antarctic krill oils on artery blood pressure in spontaneously hypertensive rats.

PubMed

Zhou, Da-Yong; Liu, Yu-Xin; Xu, Zhi-Li; Yin, Fa-Wen; Song, Liang; Wan, Xiu-Lin; Song, Yu-Kun; Zhu, Bei-Wei

2017-03-01

In recent years, there has been a noticeable increase in research on krill oil (KO) for its health benefits. However, the action of KO in lowering blood pressure (BP) has not been studied yet. Therefore the aim of this study was to assess the ability of long-term KO supplementation to lower systolic BP (SBP) in spontaneously hypertensive rats (SHRs) and Sprague Dawley (SD) rats. Compared with the blank control (BC) SHRs administered edible soybean oil, the high-dose (500 mg kg -1 body weight (BW)) KO-supplemented SHRs in the 2nd, 3rd, 4th and 5th weeks following oral administration, the mid-dose (100 mg kg -1 BW) KO-supplemented SHRs in the 4th and 5th weeks following oral administration and the low-dose (20 mg kg -1 BW) KO-supplemented SHRs in the 5th week following oral administration showed significantly lower SBP (P < 0.05). However, supplementation of KO had no significant effect on the SBP of healthy SD rats. Meanwhile, 5 weeks of KO administration significantly increased the serum levels of nitric oxide (NO) and total NO synthase of SHRs (P < 0.05). KO has an antihypertensive effect in SHRs that is associated with an NO-related mechanism. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Superoxide Stabilization and a Universal KO2 Growth Mechanism in Potassium-Oxygen Batteries.

PubMed

Wang, Wanwan; Lai, Nien-Chu; Liang, Zhuojian; Wang, Yu; Lu, Yi-Chun

2018-04-23

Rechargeable potassium-oxygen (K-O 2 ) batteries promise to provide higher round-trip efficiency and cycle life than other alkali-oxygen batteries with satisfactory gravimetric energy density (935 Wh kg -1 ). Exploiting a strong electron-donating solvent, for example, dimethyl sulfoxide (DMSO) strongly stabilizes the discharge product (KO 2 ), resulting in significant improvement in electrode kinetics and chemical/electrochemical reversibility. The first DMSO-based K-O 2 battery demonstrates a much higher energy efficiency and stability than the glyme-based electrolyte. A universal KO 2 growth model is developed and it is demonstrated that the ideal solvent for K-O 2 batteries should strongly stabilize superoxide (strong donor ability) to obtain high electrode kinetics and reversibility while providing fast oxygen diffusion to achieve high discharge capacity. This work elucidates key electrolyte properties that control the efficiency and reversibility of K-O 2 batteries. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of Perturbed Pancreatic β-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism

PubMed Central

Cochran, Blake J.; Hou, Liming; Manavalan, Anil Paul Chirackal; Moore, Benjamin M.; Tabet, Fatiha; Sultana, Afroza; Cuesta Torres, Luisa; Tang, Shudi; Shrestha, Sudichhya; Senanayake, Praween; Patel, Mili; Ryder, William J.; Bongers, Andre; Maraninchi, Marie; Wasinger, Valerie C.; Westerterp, Marit; Tall, Alan R.; Barter, Philip J.

2016-01-01

Elevated pancreatic β-cell cholesterol levels impair insulin secretion and reduce plasma insulin levels. This study establishes that low plasma insulin levels have a detrimental effect on two major insulin target tissues: adipose tissue and skeletal muscle. Mice with increased β-cell cholesterol levels were generated by conditional deletion of the ATP-binding cassette transporters, ABCA1 and ABCG1, in β-cells (β-DKO mice). Insulin secretion was impaired in these mice under basal and high-glucose conditions, and glucose disposal was shifted from skeletal muscle to adipose tissue. The β-DKO mice also had increased body fat and adipose tissue macrophage content, elevated plasma interleukin-6 and MCP-1 levels, and decreased skeletal muscle mass. They were not, however, insulin resistant. The adipose tissue expansion and reduced skeletal muscle mass, but not the systemic inflammation or increased adipose tissue macrophage content, were reversed when plasma insulin levels were normalized by insulin supplementation. These studies identify a mechanism by which perturbation of β-cell cholesterol homeostasis and impaired insulin secretion increase adiposity, reduce skeletal muscle mass, and cause systemic inflammation. They further identify β-cell dysfunction as a potential therapeutic target in people at increased risk of developing type 2 diabetes. PMID:27702832

Characterization of the insulin sensitivity of ghrelin receptor KO mice using glycemic clamps

PubMed Central

2011-01-01

Background We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity. Results Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice. Conclusions These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge. PMID:21211044

Promises and pitfalls of a Pannexin1 transgenic mouse line.

PubMed

Hanstein, Regina; Negoro, Hiromitsu; Patel, Naman K; Charollais, Anne; Meda, Paolo; Spray, David C; Suadicani, Sylvia O; Scemes, Eliana

2013-01-01

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1(tm1a(KOMP)Wtsi). Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1(f/f) mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.

Ethanol consumption in mice lacking CD14, TLR2, TLR4, or MyD88

PubMed Central

Blednov, Yuri A.; Black, Mendy; Chernis, Julia; Da Costa, Adriana; Mayfield, Jody; Harris, R. Adron

2016-01-01

Background Molecular and behavioral studies support a role for innate immune proinflammatory pathways in mediating the effects of alcohol. Increased levels of Toll-like receptors (TLRs) have been observed in animal models of alcohol consumption and in human alcoholics, and many of these TLRs signal via the MyD88-dependent pathway. We hypothesized that this pathway is involved in alcohol drinking and examined some of its key signaling components. Methods Different ethanol drinking paradigms were studied in male and female control C57BL/6J mice vs. mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We studied continuous and intermittent access two-bottle choice (2BC) and one-bottle and 2BC drinking-in-the-dark (DID) tests as well as preference for saccharin, quinine, and NaCl. Results In the 2BC continuous access test, ethanol intake decreased in male TLR2 knockout (KO) mice, and we previously reported reduced 2BC drinking in male and female CD14 KO mice. In the intermittent access 2BC test, ethanol intake decreased in CD14 KO male and female mice, whereas drinking increased in MyD88 KO male mice. In the 2BC-DID test, ethanol drinking decreased in male and female mice lacking TLR2, whereas drinking increased in MyD88 KO male mice. In the one-bottle DID test, ethanol intake decreased in female TLR2 KO mice. TLR2 KO and CD14 KO mice did not differ in saccharin preference but showed reduced preference for NaCl. MyD88 KO mice showed a slight reduction in preference for saccharin. Conclusions Deletion of key components of the MyD88-dependent pathway produced differential effects on ethanol intake by decreasing (TLR2 KO and CD14 KO) or increasing (MyD88 KO) drinking, while deletion of TLR4 had no effect. Some of the drinking effects depended on the sex of the mice and/or the ethanol-drinking model. PMID:28146272

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

PubMed Central

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

β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

PubMed Central

Kohout, Trudy A.; Lin, Fang-Tsyr; Perry, Stephen J.; Conner, David A.; Lefkowitz, Robert J.

2001-01-01

The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation. PMID:11171997

Kalrn plays key roles within and outside of the nervous system.

PubMed

Mandela, Prashant; Yankova, Maya; Conti, Lisa H; Ma, Xin-Ming; Grady, James; Eipper, Betty A; Mains, Richard E

2012-11-01

The human KALRN gene, which encodes a complex, multifunctional Rho GDP/GTP exchange factor, has been linked to cardiovascular disease, psychiatric disorders and neurodegeneration. Examination of existing Kalrn knockout mouse models has focused only on neuronal phenotypes. However, Kalirin was first identified through its interaction with an enzyme involved in the synthesis and secretion of multiple bioactive peptides, and studies in C.elegans revealed roles for its orthologue in neurosecretion. We used a broad array of tests to evaluate the effects of ablating a single exon in the spectrin repeat region of Kalrn (KalSR(KO/KO)); transcripts encoding Kalrn isoforms containing only the second GEF domain can still be produced from the single remaining functional Kalrn promoter. As expected, KalSR(KO/KO) mice showed a decrease in anxiety-like behavior and a passive avoidance deficit. No changes were observed in prepulse inhibition of acoustic startle or tests of depression-like behavior. Growth rate, parturition and pituitary secretion of growth hormone and prolactin were deficient in the KalSR(KO/KO) mice. Based on the fact that a subset of Kalrn isoforms is expressed in mouse skeletal muscle and the observation that muscle function in C.elegans requires its Kalrn orthologue, KalSR(KO/KO) mice were evaluated in the rotarod and wire hang tests. KalSR(KO/KO) mice showed a profound decrease in neuromuscular function, with deficits apparent in KalSR(+/KO) mice; these deficits were not as marked when loss of Kalrn expression was restricted to the nervous system. Pre- and postsynaptic deficits in the neuromuscular junction were observed, along with alterations in sarcomere length. Many of the widespread and diverse deficits observed both within and outside of the nervous system when expression of Kalrn is eliminated may reflect its role in secretory granule function and its expression outside of the nervous system.

Early Growth Response-1 Plays an Important Role in Ischemia-Reperfusion Injury in Lung Transplants by Regulating Polymorphonuclear Neutrophil Infiltration.

PubMed

Yamamoto, Sumiharu; Yamane, Masaomi; Yoshida, Osamu; Waki, Naohisa; Okazaki, Mikio; Matsukawa, Akihiro; Oto, Takahiro; Miyoshi, Shinichiro

2015-11-01

Early growth response-1 (Egr-1) has been shown to be a trigger-switch transcription factor that is involved in lung ischemia-reperfusion injury (IRI). Mouse lung transplants were performed in wild-type (WT) C57BL/6 and Egr1-knockout (KO) mice in the following donor → recipient combinations: WT → WT, KO → WT, WT → KO, and KO → KO to determine whether the presence of Egr-1 in the donor or recipient is the most critical factor for IRI. Pulmonary grafts were retrieved after 18 hours of ischemia after 4 hours of reperfusion. We analyzed graft function by analyzing arterial blood gas and histology in each combination and assessed the effects of Egr1 depletion on inflammatory cytokines that are regulated by Egr-1 as well on polymorphonuclear neutrophil (PMN) infiltration. Deletion of Egr1 improved pulmonary graft function in the following order of donor → recipient combinations: WT → WT < WT → KO < KO → WT < KO → KO. Polymerase chain reaction assays for Il1B, Il6, Mcp1, Mip2, Icam1, and Cox2 showed significantly lower expression levels in the KO → KO group than in the other groups. Immunohistochemistry demonstrated clear Egr-1 expression in the nuclei of pulmonary artery endothelial cells and PMN cytoplasm in the WT grafts. Flow cytometry analysis showed that Egr1 deletion reduced PMN infiltration and that the extent of reduction correlated with graft function. Both graft and recipient Egr-1 played a role in lung IRI, but the graft side contributed more to this phenomenon through regulation of PMN infiltration. Donor Egr-1 expression in pulmonary artery endothelial cells may play an important role in PMN infiltration, which results in IRI after lung transplantation.

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury

PubMed Central

Miller, Barbara A.; Hoffman, Nicholas E.; Merali, Salim; Zhang, Xue-Qian; Wang, JuFang; Rajan, Sudarsan; Shanmughapriya, Santhanam; Gao, Erhe; Barrero, Carlos A.; Mallilankaraman, Karthik; Song, Jianliang; Gu, Tongda; Hirschler-Laszkiewicz, Iwona; Koch, Walter J.; Feldman, Arthur M.; Madesh, Muniswamy; Cheung, Joseph Y.

2014-01-01

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels. PMID:24492610

Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice.

PubMed

Pavlin, T; Nagelhus, E A; Brekken, C; Eyjolfsson, E M; Thoren, A; Haraldseth, O; Sonnewald, U; Ottersen, O P; Håberg, A K

2017-01-01

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1- 13 C]glucose and [1,2- 13 C]acetate injection with ex vivo 13 C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4- 13 C]glutamate and [4- 13 C]glutamine, and percent enrichment in [4- 13 C]glutamate were detected in the α-syn KO mice. [1,2- 13 C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.

Changes in neuronal response to ischemia in retinas with genetic alterations of somatostatin receptor expression.

PubMed

Catalani, Elisabetta; Cervia, Davide; Martini, Davide; Bagnoli, Paola; Simonetti, Elisa; Timperio, Anna Maria; Casini, Giovanni

2007-03-01

Ischemia is a primary cause of neuronal death in retinal diseases. The repertoire of expressed transmitter receptors would determine the neurons' responses to ischemic damage, and peptidergic receptors may be involved. With a new in vitro model of the ischemic mouse retina, we investigated whether an altered expression of somatostatin receptors could modulate retinal responses to ischemia. We used retinas of somatostatin receptor 1 (sst(1)) knock out (KO) mice, where sst(2) are over-expressed and over-functional, and of sst(2) KO mice. TUNEL analysis of ischemic retinas showed a marked reduction of cell death in sst(1) KO retinas, while there were no differences between wild-type (WT) and sst(2) KO retinas. In addition, caspase-3 mRNA expression was also reduced in sst(1) KO as compared to WT retinas. An immunohistochemical analysis demonstrated that different cell populations responded differently to the ischemic insult, and that the persistence of some immunohistochemical markers was greater in sst(1) KO than in WT or in sst(2) KO retinas. In particular, rod bipolar cell survival was markedly improved in sst(1) KO retinas, while it was dramatically decreased in sst(2) KO retinas. Furthermore, consistent with a role of glutamate excitotoxicity in ischemia-induced neuronal death, retinal glutamate release was observed to increase under ischemic conditions, but this increase was significantly reduced in sst(1) KO retinas. These observations demonstrate that an increased presence of functional sst(2) protects against retinal ischemia, thus implementing the background for the use of sst(2) analogs in therapies of retinal diseases such as glaucoma or diabetic retinopathy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Yuri, E-mail: saito-yu@bldon.med.osaka-u.ac.jp; Shibayama, Hirohiko; Tanaka, Hirokazu

Research highlights: {yields} Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. {yields} Biological mechanisms of AM functions have not been elucidated yet. {yields} PKC{theta} , PKC{delta} and p38MAPK were more phosphorylated in AM deficient MEF cells. {yields} AM may negatively regulates PKCs and p38MAPK in MEF cells. -- Abstract: Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generatedmore » from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKC{theta}, PKC{delta}, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKC{theta}, PKC{delta}, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.« less

Interaction of growth hormone receptor/binding protein gene disruption and caloric restriction for insulin sensitivity and attenuated aging

PubMed Central

Arum, Oge; Saleh, Jamal; Boparai, Ravneet; Turner, Jeremy; Kopchick, John; Khardori, Romesh; Bartke, Andrzej

2014-01-01

The correlation of physiological sensitivity to insulin ( vis-à-vis glycemic regulation) and longevity is extensively established, creating a justifiable gerontological interest on whether insulin sensitivity is causative, or even predictive, of some or all phenotypes of slowed senescence (including longevity). The growth hormone receptor/ binding protein gene-disrupted (GHR-KO) mouse is the most extensively investigated insulin-sensitive, attenuated aging model. It was reported that, in a manner divergent from similar mutants, GHR-KO mice fail to respond to caloric restriction (CR) by altering their insulin sensitivity. We hypothesized that maximized insulin responsiveness is what causes GHR-KO mice to exhibit a suppressed survivorship response to dietary (including caloric) restriction; and attempted to refute this hypothesis by assessing the effects of CR on GHR-KO mice for varied slow-aging-associated phenotypes. In contrast to previous reports, we found GHR-KO mice on CR to be less responsive than their ad libitum (A.L.) counterparts to the hypoglycemia-inducing effects of insulin. Further, CR had negligible effects on the metabolism or cognition of GHR-KO mice. Therefore, our data suggest that the effects of CR on the insulin sensitivity of GHR-KO mice do not concur with the effects of CR on the aging of GHR-KO mice. PMID:25789159

Ensilage and bioconversion of grape pomace into fuel ethanol.

PubMed

Zheng, Yi; Lee, Christopher; Yu, Chaowei; Cheng, Yu-Shen; Simmons, Christopher W; Zhang, Ruihong; Jenkins, Bryan M; VanderGheynst, Jean S

2012-11-07

Two types of grape pomace were ensiled with eight strains of lactic acid bacteria (LAB). Both fresh grape pomace (FrGP) and fermented grape pomace (FeGP) were preserved through alcoholic fermentation but not malolactic conversion. Water leaching prior to storage was used to reduce water-soluble carbohydrates and ethanol from FrGP and FeGP, respectively, to increase malolactic conversion. Leached FeGP had spoilage after 28 days of ensilage, whereas FrGP was preserved. Dilute acid pretreatment was examined for increasing the conversion of pomace to ethanol via Escherichia coli KO11 fermentation. Dilute acid pretreatment doubled the ethanol yield from FeGP, but it did not improve the ethanol yield from FrGP. The ethanol yields from raw pomace were nearly double the yields from the ensiled pomace. For this reason, the recovery of ethanol produced during winemaking from FeGP and ethanol produced during storage of FrGP is critical for the economical conversion of grape pomace to biofuel.

Compensatory increases in tear volume and mucin levels associated with meibomian gland dysfunction caused by stearoyl-CoA desaturase-1 deficiency.

PubMed

Inaba, Takaaki; Tanaka, Yasuhisa; Tamaki, Shusaku; Ito, Tomotaka; Ntambi, James M; Tsubota, Kazuo

2018-02-20

The stearoyl-CoA desaturase (SCD) family of enzymes catalyzes monounsaturated fatty acid synthesis by inserting a cis double bond at the Δ9 position of saturated fatty acids. Disruption of these enzymes has been reported to induce a severe dry skin phenotype. Since lipid abnormalities in the meibomian glands have been associated with dry eye, we analyzed selected eye tissues contributing to tear volume and composition in genetically SCD-1-deficient mice (SCD-1 KO), including the lacrimal glands and conjunctiva. Previous histopathological analysis had revealed atrophy and loss of meibomian glands; taken together with the increased goblet cell and MUC5AC expression in the conjunctiva reported here, these findings suggest that the tear volume and mucin levels secreted are enhanced in the absence of lipid secretion as a compensatory mechanism. The expression of lipid metabolism genes in lacrimal glands was decreased in SCD1 KO mice. Thus, these results provide new pathophysiological mechanisms to pursue with regard to meibomian gland dysfunction. In addition, lack of SCD-1 causes a compensatory increase in the tear volume and mucin levels associated with changes in expression of lipid metabolism genes. These results may be useful as a new concept for dry eye treatment strategies.

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

PubMed

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

2018-05-01

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies. Copyright © 2018 Elsevier B.V. All rights reserved.

GPR21 KO mice demonstrate no resistance to high fat diet induced obesity or improved glucose tolerance.

PubMed

Wang, Jinghong; Pan, Zheng; Baribault, Helene; Chui, Danny; Gundel, Caroline; Véniant, Murielle

2016-01-01

Gpr21 KO mice generated with Gpr21 KO ES cells obtained from Deltagen showed improved glucose tolerance and insulin sensitivity when fed a high fat diet. Further mRNA expression analysis revealed changes in Rabgap1 levels and raised the possibility that Rabgap1 gene may have been modified. To assess this hypothesis a new Gpr21 KO mouse line using TALENS technology was generated. Gpr21 gene deletion was confirmed by PCR and Gpr21 and Rabgap1 mRNA expression levels were determined by RT-PCR. The newly generated Gpr21 KO mice when fed a normal or high fat diet chow did not maintain their improved metabolic phenotype. In conclusion, Rabgap1 disturbance mRNA expression levels may have contributed to the phenotype of the originally designed Gpr21 KO mice.

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse

PubMed Central

Becker, Lars; Schnee, Michael E; Niwa, Mamiko; Sun, Willy; Maxeiner, Stephan; Talaei, Sara; Kachar, Bechara; Rutherford, Mark A

2018-01-01

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon’s contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles. PMID:29328021

Effect of increasing dialysate flow rate on diffusive mass transfer of urea, phosphate and β2-microglobulin during clinical haemodialysis

PubMed Central

Bhimani, Jai P.; Ouseph, Rosemary; Ward, Richard A.

2010-01-01

Background. Diffusive clearance depends on blood and dialysate flow rates and the overall mass transfer area coefficient (KoA) of the dialyzer. Although KoA should be constant for a given dialyzer, urea KoA has been reported to vary with dialysate flow rate possibly because of improvements in flow distribution. This study examined the dependence of KoA for urea, phosphate and β2-microglobulin on dialysate flow rate in dialyzers containing undulating fibers to promote flow distribution and two different fiber packing densities. Methods. Twelve stable haemodialysis patients underwent dialysis with four different dialyzers, each used with a blood flow rate of 400 mL/min and dialysate flow rates of 350, 500 and 800 mL/min. Clearances of urea, phosphate and β2-microglobulin were measured and KoA values calculated. Results. Clearances of urea and phosphate, but not β2-microglobulin, increased significantly with increasing dialysate flow rate. However, increasing dialysate flow rate had no significant effect on KoA or Ko for any of the three solutes examined, although Ko for urea and phosphate increased significantly as the average flow velocity in the dialysate compartment increased. Conclusions. For dialyzers with features that promote good dialysate flow distribution, increasing dialysate flow rate beyond 600 mL/min at a blood flow rate of 400 mL/min is likely to have only a modest impact on dialyzer performance, limited to the theoretical increase predicted for a constant KoA. PMID:20543211

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

PubMed

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.

Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

PubMed

Mergia, Evanthia; Thieme, Manuel; Hoch, Henning; Daniil, Georgios; Hering, Lydia; Yakoub, Mina; Scherbaum, Christina Rebecca; Rump, Lars Christian; Koesling, Doris; Stegbauer, Johannes

2018-03-23

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S -nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

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

PubMed

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.

Glycolysis and Mitochondrial Respiration in Mouse LDHC-Null Sperm1

PubMed Central

Odet, Fanny; Gabel, Scott; London, Robert E.; Goldberg, Erwin; Eddy, Edward M.

2013-01-01

ABSTRACT We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect. PMID:23486916

Questionnaire survey on pediatric hypertension in Japan and Korea.

PubMed

Yim, Hyung Eun; Lee, Eun Hee; Jang, Gi Young; Yoo, Kee Hwan; Son, Chang Sung; Hong, Young Sook; Lee, Joo Won; Ito, Yuhei; Ikezumi, Yohei; Uchiyama, Makoto

2010-02-01

Hypertension (HTN) is no longer viewed as an adult disease. The purpose of the present study was to understand how hypertensive children are evaluated and managed, by surveying pediatricians in Japan and South Korea. A questionnaire was mailed to 109 Japanese (JA) and 159 Korean (KO) pediatric cardiologists, pediatric nephrologists, and other pediatricians. A total of 127 replies were received (response rate 47%). Most of respondents did not check blood pressure (BP) routinely in outpatient clinics (JA 77%, KO 88%). A mercury sphygmomanometer was the most commonly used method for BP measurements (JA 72%, KO 62%). BP treatment goals were usually set at the 95th percentile for age, gender, and height (JA 47%, KO 54%). More KO used a lower goal in children with primary HTN than JA. KO respondents preferred angiotensin-converting enzyme inhibitors (ACEI) as initial agents regardless of underlying diseases whereas JA respondents chose various medications, that is, calcium channel blockers, diuretics, and ACEI. For BP monitoring, self-monitoring was found to be most frequent in both countries (JA 80%, KO 57%). Ambulatory BP monitoring was not frequently utilized in both countries (JA 33% KO 34%). The current assessment, management and differing trends in pediatric HTN in Japan and Korea have been identified in the present study. Pediatricians should be aware of the growing implications of HTN in children.

Lack of stress responses to long-term effects of corticosterone in Caps2 knockout mice.

PubMed

Mishima, Yuriko; Shinoda, Yo; Sadakata, Tetsushi; Kojima, Masami; Wakana, Shigeharu; Furuichi, Teiichi

2015-03-10

Chronic stress is associated with anxiety and depressive disorders, and can cause weight gain. Ca(2+)-dependent activator protein for secretion 2 (CAPS2) is involved in insulin release. Caps2 knockout (KO) mice exhibit decreased body weight, reduced glucose-induced insulin release, and abnormal psychiatric behaviors. We chronically administered the stress hormone corticosterone (CORT), which induces anxiety/depressive-like behavior and normally increases plasma insulin levels, via the drinking water for 10 weeks, and we examined the stress response in KO mice. Chronic CORT exposure inhibited stress-induced serum CORT elevation in wild-type (WT) mice, but not in KO mice. Poor weight gain in CORT-treated animals was observed until week 6 in WT mice, but persisted for the entire duration of the experiment in KO mice, although there is no difference in drug*genotype interaction. Among KO mice, food consumption was unchanged, while water consumption was higher, over the duration of the experiment in CORT-treated animals, compared with untreated animals. Moreover, serum insulin and leptin levels were increased in CORT-treated WT mice, but not in KO mice. Lastly, both WT and KO mice displayed anxiety/depressive-like behavior after CORT administration. These results suggest that Caps2 KO mice have altered endocrine responses to CORT administration, while maintaining CORT-induced anxiety/depressive-like behavior.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

PubMed

Dubey, J P; Sundar, N; Kwok, O C H; Saville, W J A

2013-09-01

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals. Published by Elsevier B.V.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

PubMed

Zhang, Jifeng; Niimi, Manabu; Yang, Dongshan; Liang, Jingyan; Xu, Jie; Kimura, Tokuhide; Mathew, Anna V; Guo, Yanhong; Fan, Yanbo; Zhu, Tianqing; Song, Jun; Ackermann, Rose; Koike, Yui; Schwendeman, Anna; Lai, Liangxue; Pennathur, Subramaniam; Garcia-Barrio, Minerva; Fan, Jianglin; Chen, Y Eugene

2017-06-01

CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits. © 2017 American Heart Association, Inc.

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

PubMed

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.

Diabetes accelerates retinal ganglion cell dysfunction in mice lacking sigma receptor 1.

PubMed

Ha, Yonju; Saul, Alan; Tawfik, Amany; Zorrilla, Eric P; Ganapathy, Vadivel; Smith, Sylvia B

2012-01-01

Sigma receptor 1 (σR1) is a non-opioid transmembrane protein that may act as a molecular chaperone at the endoplasmic reticulum-mitochondrial membrane. Ligands for σR1, such as (+)-pentazocine [(+)-PTZ], confer marked retinal neuroprotection in vivo and in vitro. Recently we analyzed the retinal phenotype of mice lacking σR1 (σR1 KO) and observed normal retinal morphology and function in young mice (5-30 weeks) but diminished negative scotopic threshold responses (nSTRs), retinal ganglion cell (RGC) loss, and disruption of optic nerve axons consistent with inner retinal dysfunction by 1 year. These data led us to test the hypothesis that σR1 may be critical in forestalling chronic retinal stress; diabetes was used as the model of chronic stress. To determine whether σR1 is required for (+)-PTZ neuroprotective effects, primary RGCs isolated from wild-type (WT) and σR1 KO mice were exposed to xanthine-xanthine oxidase (10 µM:2 mU/ml) to induce oxidative stress in the presence or absence of (+)-PTZ. Cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. To assess effects of chronic stress on RGC function, diabetes was induced in 3-week C57BL/6 (WT) and σR1 KO mice, using streptozotocin to yield four groups: WT nondiabetic (WT non-DB), WT diabetic (WT-DB), σR1 KO non-DB, and σR1 KO-DB. After 12 weeks of diabetes, when mice were 15-weeks old, intraocular pressure (IOP) was recorded, electrophysiologic testing was performed (including detection of nSTRs), and the number of RGCs was counted in retinal histological sections. In vitro studies showed that (+)-PTZ could not prevent oxidative stress-induced death of RGCs harvested from σR1 KO mice but afforded robust protection against death of RGCs harvested from WT mice. In the studies of chronic stress induced by diabetes, the IOP measured in the four mouse groups was within the normal range; however, there was a significant increase in the IOP of σR1 KO-DB mice (16 ± 0.5 mmHg) compared to the other groups tested (σR1 KO non-DB, WT non-DB, WT-DB: ~12 ± 0.6 mmHg). Regarding electrophysiologic testing, the nSTRs of σR1 KO non-DB mice were similar to WT non-DB mice at 15 weeks; however, they were significantly lower in σR1 KO-DB mice (5 ± 1 µV) compared to the other groups, including, notably, σR1 KO-nonDB (12±2 µV). As expected, the number of RGCs in σR1 KO non-DB mice was similar to WT non-DB mice at 15 weeks, but under chronic stress of diabetes there were fewer RGCs in retinas of σR1 KO-DB mice. This is the first report showing unequivocally that the neuroprotective effects of (+)-PTZ require σR1. σR1 KO mice show normal retinal structure and function at young ages; however, when subjected to the chronic stress of diabetes, there is an acceleration of retinal functional deficits in σR1 KO mice such that ganglion cell dysfunction is observed at a much earlier age than nondiabetic σR1 KO mice. The data support the hypothesis that σR1 plays a key role in modulating retinal stress and may be an important target for retinal disease.

Serotonin Reuptake Transporter Deficiency Modulates the Acute Thermoregulatory and Locomotor Activity Response to 3,4-(±)-Methylenedioxymethamphetamine, and Attenuates Depletions in Serotonin Levels in SERT-KO Rats

PubMed Central

Lizarraga, Lucina E.; Phan, Andy V.; Cholanians, Aram B.; Herndon, Joseph M.; Lau, Serrine S.; Monks, Terrence J.

2014-01-01

3,4-(±)-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT), inducing thermogenesis and hyperactivity (5-HT syndrome). The long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to 5-HT nerve terminals. MDMA toxicity is in part mediated by an ability to inhibit the presynaptic 5-HT reuptake transporter (SERT). Using a SERT-knockout (SERT-KO) rat model, we determined the impact of SERT deficiency on thermoregulation, locomotor activity, and neurotoxicity in SERT-KO or Wistar-based wild-type (WT) rats exposed to MDMA. WT and SERT-KO animals exhibited the highest thermogenic responses to MDMA (four times 10 mg/kg, sc at 12 h intervals) during the diurnal (first and third) doses according to peak body temperature and area under the curve (∑°C × h) analysis. Although no differences in peak body temperature were observed between MDMA-treated WT and SERT-KO animals, ∑°C × h following the first MDMA dose was reduced in SERT-KO rats. Exposure to a single dose of MDMA stimulated horizontal velocity in both WT and SERT-KO rats, however, this effect was delayed and attenuated in the KO animals. Finally, SERT-KO rats were insensitive to MDMA-induced long-term (7 days) depletions in 5-HT and its metabolite, 5-hydroxyindole acetic acid, in both cortex and striatum. In conclusion, SERT deficiency modulated MDMA-mediated thermogenesis, hyperactivity and neurotoxicity in KO rats. The data confirm that the SERT is essential for the manifestation of the acute and long-term toxicities of MDMA. PMID:24595820

Production of Mice Deficient in Genes for Interleukin (IL)-1α, IL-1β, IL-1α/β, and IL-1 Receptor Antagonist Shows that IL-1β Is Crucial in Turpentine-induced Fever Development and Glucocorticoid Secretion

PubMed Central

Horai, Reiko; Asano, Masahide; Sudo, Katsuko; Kanuka, Hirotaka; Suzuki, Masatoshi; Nishihara, Masugi; Takahashi, Michio; Iwakura, Yoichiro

1998-01-01

Interleukin (IL)-1 is a major mediator of inflammation and exerts pleiotropic effects on the neuro-immuno-endocrine system. To elucidate pathophysiological roles of IL-1, we have first produced IL-1α/β doubly deficient (KO) mice together with mice deficient in either the IL-1α, IL-1β, or IL-1 receptor antagonist (IL-1ra) genes. These mice were born healthy, and their growth was normal except for IL-1ra KO mice, which showed growth retardation after weaning. Fever development upon injection with turpentine was suppressed in IL-1β as well as IL-1α/β KO mice, but not in IL-1α KO mice, whereas IL-1ra KO mice showed an elevated response. At this time, expression of IL-1β mRNA in the diencephalon decreased 1.5-fold in IL-1α KO mice, whereas expression of IL-1α mRNA decreased >30-fold in IL-1β KO mice, suggesting mutual induction between IL-1α and IL-1β. This mutual induction was also suggested in peritoneal macrophages stimulated with lipopolysaccharide in vitro. In IL-1β KO mice treated with turpentine, the induction of cyclooxygenase-2 (EC 1.14.99.1) in the diencephalon was suppressed, whereas it was enhanced in IL-1ra KO mice. We also found that glucocorticoid induction 8 h after turpentine treatment was suppressed in IL-1β but not IL-1α KO mice. These observations suggest that IL-1β but not IL-1α is crucial in febrile and neuro-immuno-endocrine responses, and that this is because IL-1α expression in the brain is dependent on IL-1β. The importance of IL-1ra both in normal physiology and under stress is also suggested. PMID:9565638

Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta, and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever development and glucocorticoid secretion.

PubMed

Horai, R; Asano, M; Sudo, K; Kanuka, H; Suzuki, M; Nishihara, M; Takahashi, M; Iwakura, Y

1998-05-04

Interleukin (IL)-1 is a major mediator of inflammation and exerts pleiotropic effects on the neuro-immuno-endocrine system. To elucidate pathophysiological roles of IL-1, we have first produced IL-1alpha/beta doubly deficient (KO) mice together with mice deficient in either the IL-1alpha, IL-1beta, or IL-1 receptor antagonist (IL-1ra) genes. These mice were born healthy, and their growth was normal except for IL-1ra KO mice, which showed growth retardation after weaning. Fever development upon injection with turpentine was suppressed in IL-1beta as well as IL-1alpha/beta KO mice, but not in IL-1alpha KO mice, whereas IL-1ra KO mice showed an elevated response. At this time, expression of IL-1beta mRNA in the diencephalon decreased 1.5-fold in IL-1alpha KO mice, whereas expression of IL-1alpha mRNA decreased >30-fold in IL-1beta KO mice, suggesting mutual induction between IL-1alpha and IL-1beta. This mutual induction was also suggested in peritoneal macrophages stimulated with lipopolysaccharide in vitro. In IL-1beta KO mice treated with turpentine, the induction of cyclooxygenase-2 (EC 1.14.99.1) in the diencephalon was suppressed, whereas it was enhanced in IL-1ra KO mice. We also found that glucocorticoid induction 8 h after turpentine treatment was suppressed in IL-1beta but not IL-1alpha KO mice. These observations suggest that IL-1beta but not IL-1alpha is crucial in febrile and neuro-immuno-endocrine responses, and that this is because IL-1alpha expression in the brain is dependent on IL-1beta. The importance of IL-1ra both in normal physiology and under stress is also suggested.

Bicarbonate promotes BK-α/β4-mediated K excretion in the renal distal nephron

PubMed Central

Cornelius, Ryan J.; Wen, Donghai; Hatcher, Lori I.

2012-01-01

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-β4 in acid/base-transporting intercalated cells (IC), we determined whether BK-β4 knockout mice (β4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-β4 expression increased (Western blot). When WT and β4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and β4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with β4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and β4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in β-IC, comparing WT and β4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in β4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of β4KO. When WT on LNaHK were given NH4Cl in the drinking water, K excretion was reduced to the magnitude of β4KO on LNaHK. These results show that WT, but not β4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/β4-mediated K secretion is promoted by bicarbonaturia. PMID:22993067

Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

PubMed

Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A; Sieving, Paul A

2012-09-19

Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

Koala retroviruses: characterization and impact on the life of koalas.

PubMed

Denner, Joachim; Young, Paul R

2013-10-23

Koala retroviruses (KoRV) have been isolated from wild and captive koalas in Australia as well as from koala populations held in zoos in other countries. They are members of the genus Gammaretrovirus, are most closely related to gibbon ape leukemia virus (GaLV), feline leukemia virus (FeLV) and porcine endogenous retrovirus (PERV) and are likely the result of a relatively recent trans-species transmission from rodents or bats. The first KoRV to be isolated, KoRV-A, is widely distributed in the koala population in both integrated endogenous and infectious exogenous forms with evidence from museum specimens older than 150 years, indicating a relatively long engagement with the koala population. More recently, additional subtypes of KoRV that are not endogenized have been identified based on sequence differences and host cell receptor specificity (KoRV-B and KoRV-J). A specific association with fatal lymphoma and leukemia has been recently suggested for KoRV-B. In addition, it has been proposed that the high viral loads found in many animals may lead to immunomodulation resulting in a higher incidence of diseases such as chlamydiosis. Although the molecular basis of this immunomodulation is still unclear, purified KoRV particles and a peptide corresponding to a highly conserved domain in the envelope protein have been shown to modulate cytokine expression in vitro, similar to that induced by other gammaretroviruses. While much is still to be learned, KoRV induced lymphoma/leukemia and opportunistic disease arising as a consequence of immunomodulation are likely to play an important role in the stability of koala populations both in the wild and in captivity.

Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

PubMed

Tanaka, Makoto; Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Hotta, Yuma; Toyokawa, Yuki; Ushiroda, Chihiro; Hirai, Yasuko; Aoi, Wataru; Higashimura, Yasuki; Mizushima, Katsura; Okayama, Tetsuya; Katada, Kazuhiro; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Itoh, Yoshito

2018-03-01

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4 + T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4 + T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4 + T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Knockout of arsenic (+3 oxidation state) methyltransferase is associated with adverse metabolic phenotype in mice: the role of sex and arsenic exposure.

PubMed

Douillet, Christelle; Huang, Madelyn C; Saunders, R Jesse; Dover, Ellen N; Zhang, Chongben; Stýblo, Miroslav

2017-07-01

Susceptibility to toxic effects of inorganic arsenic (iAs) depends, in part, on efficiency of iAs methylation by arsenic (+3 oxidation state) methyltransferase (AS3MT). As3mt-knockout (KO) mice that cannot efficiently methylate iAs represent an ideal model to study the association between iAs metabolism and adverse effects of iAs exposure, including effects on metabolic phenotype. The present study compared measures of glucose metabolism, insulin resistance and obesity in male and female wild-type (WT) and As3mt-KO mice during a 24-week exposure to iAs in drinking water (0.1 or 1 mg As/L) and in control WT and As3mt-KO mice drinking deionized water. Results show that effects of iAs exposure on fasting blood glucose (FBG) and glucose tolerance in either WT or KO mice were relatively minor and varied during the exposure. The major effects were associated with As3mt KO. Both male and female control KO mice had higher body mass with higher percentage of fat than their respective WT controls. However, only male KO mice were insulin resistant as indicated by high FBG, and high plasma insulin at fasting state and 15 min after glucose challenge. Exposure to iAs increased fat mass and insulin resistance in both male and female KO mice, but had no significant effects on body composition or insulin resistance in WT mice. These data suggest that As3mt KO is associated with an adverse metabolic phenotype that is characterized by obesity and insulin resistance, and that the extent of the impairment depends on sex and exposure to iAs, including exposure to iAs from mouse diet.

Glial dysfunction in parkin null mice: effects of aging.

PubMed

Solano, Rosa M; Casarejos, Maria J; Menéndez-Cuervo, Jamie; Rodriguez-Navarro, Jose A; García de Yébenes, Justo; Mena, Maria A

2008-01-16

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals, and abnormal neurotransmitter release. The role of glia in parkin deficiency is little known. We cultured midbrain glia from wild-type (WT) and parkin knock-out (PK-KO) mice. After 18-20 d in vitro, PK-KO glial cultures had less astrocytes, more microglia, reduced proliferation, and increased proapoptotic protein expression. PK-KO glia had greater levels of intracellular glutathione (GSH), increased mRNA expression of the GSH-synthesizing enzyme gamma-glutamylcysteine synthetase, and greater glutathione S-transferase and lower glutathione peroxidase activities than WT. The reverse happened in glia cultured in serum-free defined medium (EF12) or in old cultures. PK-KO glia was more susceptible than WT to transference to EF12 or neurotoxins (1-methyl-4-phenylpyridinium, blockers of GSH synthesis or catalase, inhibitors of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3 kinases), aging of the culture, or combination of these insults. PK-KO glia was less susceptible than WT to Fe2+ plus H2O2 and less responsive to protection by deferoxamine. Old WT glia increased the expression of heat shock protein 70, but PK-KO did not. Glia conditioned medium (GCM) from PK-KO was less neuroprotective and had lower levels of GSH than WT. GCM from WT increased the levels of dopamine markers in midbrain neuronal cultures transferred to EF12 more efficiently than GCM from PK-KO, and the difference was corrected by supplementation with GSH. PK-KO-GCM was a less powerful suppressor of apoptosis and microglia in neuronal cultures. Our data prove that abnormal glial function is critical in parkin mutations, and its role increases with aging.

Central representation of postingestive chemosensory cues in mice that lack the ability to taste.

PubMed

Stratford, Jennifer M; Finger, Thomas E

2011-06-22

The gustatory nerves of mice lacking P2X2 and P2X3 purinergic receptor subunits (P2X-dblKO) are unresponsive to taste stimulation (Finger et al., 2005). Surprisingly, P2X-dblKO mice show residual behavioral responses to concentrated tastants, presumably via postingestive detection. Therefore, the current study tested whether postingestive signaling is functional in P2X-dblKO mice and if so, whether it activates the primary viscerosensory nucleus of the medulla, the nucleus of the solitary tract (nTS). Like WT animals, P2X-dblKO mice learned to prefer a flavor paired with 150 mm monosodium glutamate (MSG) over a flavor paired with water. This preference shows that, even in the absence of taste sensory input, postingestive cues are detected and associated with a flavor in P2X-dblKO mice. MSG-evoked neuronal activation in the nTS was measured by expression of the immediate early gene c-Fos [c-Fos-like immunoreactivity (Fos-LI)]. In rostral, gustatory nTS, P2X-dblKO animals, unlike WT animals, showed no taste quality-specific labeling of neurons. Furthermore, MSG-evoked Fos-LI was significantly less in P2X-dblKO mice compared with WT animals. In contrast, in more posterior, viscerosensory nTS, MSG-induced Fos-LI was similar in WT and P2X-dblKO mice. Together, these results suggest that P2X-dblKO mice can form preferences based on postingestive cues and that postingestive detection of MSG does not rely on the same purinergic signaling that is crucial for taste.

RECG Maintains Plastid and Mitochondrial Genome Stability by Suppressing Extensive Recombination between Short Dispersed Repeats

PubMed Central

Odahara, Masaki; Masuda, Yuichi; Sato, Mayuko; Wakazaki, Mayumi; Harada, Chizuru; Toyooka, Kiminori; Sekine, Yasuhiko

2015-01-01

Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8–79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12–63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions. PMID:25769081

Protective role of somatostatin receptor 2 against retinal degeneration in response to hypoxia.

PubMed

Dal Monte, Massimo; Latina, Valentina; Cupisti, Elena; Bagnoli, Paola

2012-05-01

In mouse retinal explants, octreotide, a somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor 2 (sst(2)) agonist, prevents the hypoxia-induced vascular endothelial growth factor upregulation. In mice with oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity, either sst(2) overexpression or octreotide have been found to limit hypoxia-induced angiogenic processes. Here, we investigated whether sst(2) influences retinal degeneration in response to hypoxia in wild-type (WT), sst(1)- and sst(2)-knockout (KO) mice. In retinal explants, we determined the role of sst(2) on apoptotic signals. In control condition, caspase-3 activity and the Bax/Bcl-2 ratio were lower in sst(1)-KO than in WT, but higher in sst(2)-KO than in WT retinas. In all strains, a comparable increase in caspase-3 activity and the Bax/Bcl-2 ratio was observed after hypoxia. The hypoxia-induced increase in apoptotic signals was recovered by octreotide in both WT and sst(1)-KO retinas. To investigate the role of sst(2) on retinal function, we recorded electroretinogram (ERG) in response to light flashes in OIR mice. ERG responses did not differ between WT and KO mice with the exception of oscillatory potentials (OPs) which, in sst(1)-KO mice, displayed much larger amplitude. In all strains, hypoxia drastically reduced a-, b-waves and OPs. In both WT and sst(1)-KO mice, octreotide recovered a- and b-waves, but did not recover OPs in sst(1)-KO mice. Neither apoptotic signals nor ERG was affected by octreotide in sst(2)-KO mice. These results show that sst(2) may protect retinal cells from hypoxia, thus implementing the background to establish potential pharmacological targets based on sst(2) pharmacology.

Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: A sex-specific gene profiling analysis.

PubMed

Wang, Hao; Sun, Xuming; Chou, Jeff; Lin, Marina; Ferrario, Carlos M; Zapata-Sudo, Gisele; Groban, Leanne

2017-08-01

Activation of G protein-coupled estrogen receptor (GPER) by its agonist, G1, protects the heart from stressors such as pressure-overload, ischemia, a high-salt diet, estrogen loss, and aging, in various male and female animal models. Due to nonspecific effects of G1, the exact functions of cardiac GPER cannot be concluded from studies using systemic G1 administration. Moreover, global knockdown of GPER affects glucose homeostasis, blood pressure, and many other cardiovascular-related systems, thereby confounding interpretation of its direct cardiac actions. We generated a cardiomyocyte-specific GPER knockout (KO) mouse model to specifically investigate the functions of GPER in cardiomyocytes. Compared to wild type mice, cardiomyocyte-specific GPER KO mice exhibited adverse alterations in cardiac structure and impaired systolic and diastolic function, as measured by echocardiography. Gene deletion effects on left ventricular dimensions were more profound in male KO mice compared to female KO mice. Analysis of DNA microarray data from isolated cardiomyocytes of wild type and KO mice revealed sex-based differences in gene expression profiles affecting multiple transcriptional networks. Gene Set Enrichment Analysis (GSEA) revealed that mitochondrial genes are enriched in GPER KO females, whereas inflammatory response genes are enriched in GPER KO males, compared to their wild type counterparts of the same sex. The cardiomyocyte-specific GPER KO mouse model provides us with a powerful tool to study the functions of GPER in cardiomyocytes. The gene expression profiles of the GPER KO mice provide foundational information for further study of the mechanisms underlying sex-specific cardioprotection by GPER. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

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

IL-4 Knock out Mice Display Anxiety-like Behavior

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-07-01

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

The specificity of cortical region KO to depth structure.

PubMed

Tyler, Christopher W; Likova, Lora T; Kontsevich, Leonid L; Wade, Alex R

2006-03-01

Functional MRI studies have identified a cortical region designated as KO between retinotopic areas V3A/B and motion area V5 in human cortex as particularly responsive to motion-defined or kinetic borders. To determine the response of the KO region to more general aspects of structure, we used stereoscopic depth borders and disparate planes with no borders, together with three stimulus types that evoked no depth percept: luminance borders, line contours and illusory phase borders. Responses to these stimuli in the KO region were compared with the responses in retinotopically defined areas that have been variously associated with disparity processing in neurophysiological and fMRI studies. The strongest responses in the KO region were to stimuli evoking perceived depth structure from either disparity or motion cues, but it showed negligible responses either to luminance-based contour stimuli or to edgeless disparity stimuli. We conclude that the region designated as KO is best regarded as a primary center for the generic representation of depth structure rather than any kind of contour specificity.

Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis

PubMed Central

Midura-Kiela, Monica T.; Ramalingam, Rajalakshmy; Larmonier, Claire B.; Chase, John H.; Caporaso, J. Gregory; Besselsen, David G.; Ghishan, Fayez K.; Kiela, Pawel R.

2016-01-01

Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community. PMID:27050757

MiR-143/145 deficiency attenuates the progression of atherosclerosis in Ldlr-/-mice.

PubMed

Sala, Federica; Aranda, Juan F; Rotllan, Noemi; Ramírez, Cristina M; Aryal, Binod; Elia, Leonardo; Condorelli, Gianluigi; Catapano, Alberico Luigi; Fernández-Hernando, Carlos; Norata, Giuseppe Danilo

2014-10-01

The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Ldlr-/- and Ldlr-/--miR-143/145-/- (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. We further confirmed the direct regulation of miR-145 on ABCA1 expression by qRT-PCR, Western blotting and 3'UTR-luciferase reporter assays. In summary, miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.

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

PubMed Central

Hasen, Nina S.; Gammie, Stephen C.

2010-01-01

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. PMID:21070815

Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

PubMed Central

Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

2016-01-01

Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

The Inhibitor Ko143 Is Not Specific for ABCG2.

PubMed

Weidner, Lora D; Zoghbi, Sami S; Lu, Shuiyu; Shukla, Suneet; Ambudkar, Suresh V; Pike, Victor W; Mulder, Jan; Gottesman, Michael M; Innis, Robert B; Hall, Matthew D

2015-09-01

Imaging ATP-binding cassette (ABC) transporter activity in vivo with positron emission tomography requires both a substrate and a transporter inhibitor. However, for ABCG2, there is no inhibitor proven to be specific to that transporter alone at the blood-brain barrier. Ko143 [[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4- b]indole-3-propanoic acid 1,1-dimethylethyl ester], a nontoxic analog of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although its specificity in mouse and human systems is unclear. This study examined the selectivity of Ko143 using human embryonic kidney cell lines transfected with ABCG2, ABCB1, or ABCC1 in several in vitro assays. The stability of Ko143 in rat plasma was measured using high performance liquid chromatography. Our results show that, in addition to being a potent inhibitor of ABCG2, at higher concentrations (≥1 μM) Ko143 also has an effect on the transport activity of both ABCB1 and ABCC1. Furthermore, Ko143 was found to be unstable in rat plasma. These findings indicate that Ko143 lacks specificity for ABCG2 and this should be taken into consideration when using Ko143 for both in vitro and in vivo experiments. U.S. Government work not protected by U.S. copyright.

Loss of TGF-β signaling in osteoblasts increases basic-FGF and promotes prostate cancer bone metastasis.

PubMed

Meng, Xiangqi; Vander Ark, Alexandra; Daft, Paul; Woodford, Erica; Wang, Jie; Madaj, Zachary; Li, Xiaohong

2018-04-01

TGF-β plays a central role in prostate cancer (PCa) bone metastasis, and it is crucial to understand the bone cell-specific role of TGF-β signaling in this process. Thus, we used knockout (KO) mouse models having deletion of the Tgfbr2 gene specifically in osteoblasts (Tgfbr2 Col1CreERT KO) or in osteoclasts (Tgfbr2 LysMCre KO). We found that PCa-induced bone lesion development was promoted in the Tgfbr2 Col1CreERT KO mice, but was inhibited in the Tgfbr2 LysMCre KO mice, relative to their respective control Tgfbr2 FloxE2 littermates. Since metastatic PCa cells attach to osteoblasts when colonized in the bone microenvironment, we focused on the mechanistic studies using the Tgfbr2 Col1CreERT KO mouse model. We found that bFGF was upregulated in osteoblasts from PC3-injected tibiae of Tgfbr2 Col1CreERT KO mice and correlated with increased tumor cell proliferation, angiogenesis, amounts of cancer-associated fibroblasts and osteoclasts. In vitro studies showed that osteoblastogenesis was inhibited, osteoclastogenesis was stimulated, but PC3 viability was not affected, by bFGF treatments. Lastly, the increased PC3-induced bone lesions in Tgfbr2 Col1CreERT KO mice were significantly attenuated by blocking bFGF using neutralizing antibody, suggesting bFGF is a promising target inhibiting bone metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Contribution of Invariant Natural Killer T Cells to Skin Wound Healing.

PubMed

Tanno, Hiromasa; Kawakami, Kazuyoshi; Ritsu, Masae; Kanno, Emi; Suzuki, Aiko; Kamimatsuno, Rina; Takagi, Naoyuki; Miyasaka, Tomomitsu; Ishii, Keiko; Imai, Yoshimichi; Maruyama, Ryoko; Tachi, Masahiro

2015-12-01

In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

PubMed

Gibson, Monica Prasad; Zhu, Qinglin; Wang, Suzhen; Liu, Qilin; Liu, Ying; Wang, Xiaofang; Yuan, Baozhi; Ruest, L Bruno; Feng, Jian Q; D'Souza, Rena N; Qin, Chunlin; Lu, Yongbo

2013-03-08

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Directionally Efficient Robust Estimators of Location Via Exponential Embedding.

DTIC Science & Technology

1983-05-01

20 25 25 40 40 45 47.5 JOH 40 20 20 22.5 TAK 20 10 15 17.5 JAE 20 20 25 25 HG1 20 30 35 37.5 HG2 40 30 35 37.5 largest j for which the estimator is...SK(50) ,FY(2,50) COMPLEX ZSM ,ZLG 0 LOGICAL GCASE COMMON DT1 .RN,DRS,DRK EXTERNAL F1,F2 DATA SQRT8/2.828427125D0/4 RN=DBLE(N) NHALF=N/2 NHP1-NHALF+l M...35j KO=KO-l 6O TO 30 35 IF((IFLAG .EQ. 0) .AND. (KO .EQ. NHALF)) THEN THETA-XDAR GO TO 700 END IF s1.0.0 DO 50 X=KO,1,-1 *50 S1=XDAR-X(I).Sl K-Ko

Fat-specific Dicer deficiency accelerates aging and mitigates several effects of dietary restriction in mice.

PubMed

Reis, Felipe C G; Branquinho, Jéssica L O; Brandão, Bruna B; Guerra, Beatriz A; Silva, Ismael D; Frontini, Andrea; Thomou, Thomas; Sartini, Loris; Cinti, Saverio; Kahn, C Ronald; Festuccia, William T; Kowaltowski, Alicia J; Mori, Marcelo A

2016-06-01

Aging increases the risk of type 2 diabetes, and this can be prevented by dietary restriction (DR). We have previously shown that DR inhibits the downregulation of miRNAs and their processing enzymes - mainly Dicer - that occurs with aging in mouse white adipose tissue (WAT). Here we used fat-specific Dicer knockout mice (AdicerKO) to understand the contributions of adipose tissue Dicer to the metabolic effects of aging and DR. Metabolomic data uncovered a clear distinction between the serum metabolite profiles of Lox control and AdicerKO mice, with a notable elevation of branched-chain amino acids (BCAA) in AdicerKO. These profiles were associated with reduced oxidative metabolism and increased lactate in WAT of AdicerKO mice and were accompanied by structural and functional changes in mitochondria, particularly under DR. AdicerKO mice displayed increased mTORC1 activation in WAT and skeletal muscle, where Dicer expression is not affected. This was accompanied by accelerated age-associated insulin resistance and premature mortality. Moreover, DR-induced insulin sensitivity was abrogated in AdicerKO mice. This was reverted by rapamycin injection, demonstrating that insulin resistance in AdicerKO mice is caused by mTORC1 hyperactivation. Our study evidences a DR-modulated role for WAT Dicer in controlling metabolism and insulin resistance.

Inactivation of adipose angiotensinogen reduces adipose tissue macrophages and increases metabolic activity.

PubMed

LeMieux, Monique J; Ramalingam, Latha; Mynatt, Randall L; Kalupahana, Nishan S; Kim, Jung Han; Moustaïd-Moussa, Naïma

2016-02-01

The adipose renin-angiotensin system (RAS) has been linked to obesity-induced inflammation, though mechanisms are not completely understood. In this study, adipose-specific angiotensinogen knockout mice (Agt-KO) were generated to determine whether Agt inactivation reduces inflammation and alters the metabolic profile of the Agt-KO mice compared to wild-type (WT) littermates. Adipose tissue-specific Agt-KO mice were created using the Cre-LoxP system with both Agt-KO and WT littermates fed either a low-fat or high-fat diet to assess metabolic changes. White adipose tissue was used for gene/protein expression analyses and WAT stromal vascular cells for metabolic extracellular flux assays. No significant differences were observed in body weight or fat mass between both genotypes on either diet. However, improved glucose clearance was observed in Agt-KO compared to WT littermates, consistent with higher expression of genes involved in insulin signaling, glucose transport, and fatty acid metabolism. Furthermore, Agt inactivation reduced total macrophage infiltration in Agt-KO mice fed both diets. Lastly, stroma vascular cells from Agt-KO mice revealed higher metabolic activity compared to WT mice. These findings indicate that adipose-specific Agt inactivation leads to reduced adipose inflammation and increased glucose tolerance mediated in part via increased metabolic activity of adipose cells. © 2015 The Obesity Society.

Fat-specific Dicer deficiency accelerates aging and mitigates several effects of dietary restriction in mice

PubMed Central

Reis, Felipe C. G.; Branquinho, Jéssica L. O.; Brandão, Bruna B.; Guerra, Beatriz A.; Silva, Ismael D.; Frontini, Andrea; Thomou, Thomas; Sartini, Loris; Cinti, Saverio; Kahn, C. Ronald; Festuccia, William T.; Kowaltowski, Alicia J.; Mori, Marcelo A.

2016-01-01

Aging increases the risk of type 2 diabetes, and this can be prevented by dietary restriction (DR). We have previously shown that DR inhibits the downregulation of miRNAs and their processing enzymes - mainly Dicer - that occurs with aging in mouse white adipose tissue (WAT). Here we used fat-specific Dicer knockout mice (AdicerKO) to understand the contributions of adipose tissue Dicer to the metabolic effects of aging and DR. Metabolomic data uncovered a clear distinction between the serum metabolite profiles of Lox control and AdicerKO mice, with a notable elevation of branched-chain amino acids (BCAA) in AdicerKO. These profiles were associated with reduced oxidative metabolism and increased lactate in WAT of AdicerKO mice and were accompanied by structural and functional changes in mitochondria, particularly under DR. AdicerKO mice displayed increased mTORC1 activation in WAT and skeletal muscle, where Dicer expression is not affected. This was accompanied by accelerated age-associated insulin resistance and premature mortality. Moreover, DR-induced insulin sensitivity was abrogated in AdicerKO mice. This was reverted by rapamycin injection, demonstrating that insulin resistance in AdicerKO mice is caused by mTORC1 hyperactivation. Our study evidences a DR-modulated role for WAT Dicer in controlling metabolism and insulin resistance. PMID:27241713

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

PubMed Central

Nakamura, Toru; Sato, Asako; Kitsukawa, Takashi; Momiyama, Toshihiko; Yamamori, Tetsuo; Sasaoka, Toshikuni

2014-01-01

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 5 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 and mutant mice. PMID:25076876

Sex differences in the development of diabetes in mice with deleted wolframin (Wfs1) gene.

PubMed

Noormets, K; Kõks, S; Muldmaa, M; Mauring, L; Vasar, E; Tillmann, V

2011-05-01

Wolfram syndrome, caused by mutations in the wolframin (Wfs1) gene, is characterised by juvenile-onset diabetes mellitus, progressive optic atrophy, diabetes insipidus and deafness. Diabetes tend to start earlier in boys. This study investigated sex differences in longitudinal changes in blood glucose concentration (BGC) in wolframin-deficient mice (Wfs1KO) and compared their plasma proinsulin and insulin levels with those of wild-type (wt) mice. Non-fasting BGC was measured weekly in 42 (21 males) mice from both groups at nine weeks of age. An intraperitoneal glucose tolerance test (IPGTT) was conducted at the 30 (th) week and plasma insulin, c-peptide and proinsulin levels were measured at the 32 (nd) week. At the 32 (nd) week, Wfs1KO males had increased BGC compared to wt males (9.40±0.60 mmol/l vs. 7.91±0.20 mmol/l; p<0.05). The opposite tendency was seen in females. Both male and female Wfs1KO mice had impaired glucose tolerance on IPGTT. Wfs1KO males had significantly lower mean plasma insulin levels than wt males (57.78±1.80 ng/ml vs. 69.42±3.06 ng/ml; p<0.01) and Wfs1KO females (70.30±4.42 ng/ml; p<0.05). Wfs1KO males had a higher proinsulin/insulin ratio than wt males (0.09±0.02 vs. 0.05±0.01; p=0.05) and Wfs1KO females (0.04±0.01; p<0.05). Plasma c-peptide levels in males were lower in Wfs1KO males (mean 55.3±14.0 pg/ml vs. 112.7±21.9 pg/ml; p<0.05). Male Wfs1KO mice had a greater risk of developing diabetes than female Wfs1KO mice. Low plasma insulin concentration with an increased proinsulin/insulin ratio in Wfs1KO males indicates possible disturbances in converting proinsulin to insulin which in long-term may lead to insulin deficiency. Further investigation is needed to clarify the mechanism for the sex differences in the development of diabetes in Wolfram syndrome. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

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

PubMed

Nakamura, Toru; Sato, Asako; Kitsukawa, Takashi; Momiyama, Toshihiko; Yamamori, Tetsuo; Sasaoka, Toshikuni

2014-01-01

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 5 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 and mutant mice.

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

PubMed Central

Haack, Nicole; Durry, Simone; Kafitz, Karl W.; Chesler, Mitchell; Rose, Christine R.

2015-01-01

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called “concentric” ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations. PMID:26381747

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-05-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response.

PubMed

Gri, Giorgia; Gallo, Elena; Di Carlo, Emma; Musiani, Piero; Colombo, Mario P

2003-01-01

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

Cerebral Cortex Hyperthyroidism of Newborn Mct8-Deficient Mice Transiently Suppressed by Lat2 Inactivation

PubMed Central

Núñez, Bárbara; Martínez de Mena, Raquel; Obregon, Maria Jesus; Font-Llitjós, Mariona; Nunes, Virginia; Palacín, Manuel; Dumitrescu, Alexandra M.; Morte, Beatriz; Bernal, Juan

2014-01-01

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2 -/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3′-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3′-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development. PMID:24819605

Cerebral cortex hyperthyroidism of newborn mct8-deficient mice transiently suppressed by lat2 inactivation.

PubMed

Núñez, Bárbara; Martínez de Mena, Raquel; Obregon, Maria Jesus; Font-Llitjós, Mariona; Nunes, Virginia; Palacín, Manuel; Dumitrescu, Alexandra M; Morte, Beatriz; Bernal, Juan

2014-01-01

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2-/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3'-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3'-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb.

PubMed

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-05-27

Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb

PubMed Central

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-01-01

ABSTRACT Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb. PMID:28340332

SOCS3 Deletion in T-Lymphocytes Suppresses Development of Chronic Ocular Inflammation Via Up-regulation of CTLA-4 and Expansion of Regulatory T cells

PubMed Central

Yu, Cheng-Rong; Kim, Sung-Hye; Mahdi, Rashid M.; Egwuagu, Charles E.

2013-01-01

Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of JAK/STAT pathway and SOCS3 contributes to host immunity by regulating the intensity/duration of cytokine signals and inflammatory responses. Mice with Socs3 deletion in myeloid cells exhibit enhanced STAT3-signaling, expansion of Th1 and Th17 cells and developed severe experimental autoimmune encephalomyelitis (EAE). Interestingly, development of the unique IL-17/IFN-γ-double producing (Th17/IFN-γ and Tc17/IFN-γ) subsets that exhibit strong cytotoxic activities and associated with pathogenesis of several autoimmune diseases, has recently been shown to depend on epigenetic suppression of SOCS3 expression, further suggesting involvement of SOCS3 in autoimmunity and tumor immunity. In this study, we generated mice with Socs3 deletion in CD4 T cell compartment (CD4-SOCS3KO) to determine in vivo effects of the loss of Socs3 in the T cell-mediated autoimmune disease, experimental autoimmune uveitis (EAU). In contrast to the exacerbation of EAE in myeloid-specific SOCS3-deleted mice, CD4-SOCS3KO mice were protected from acute and chronic uveitis. Protection from EAU correlated with enhanced expression of CTLA4 and expansion of IL-10 producing Tregs with augmented suppressive activities. We further show that SOCS3 interacts with CTLA4 and negatively regulates CTLA4 levels in T cells, providing mechanistic explanation for the expansion of Tregs in CD4-SOCS3 during EAU. Contrary to in vitro epigenetic studies, Th17/IFN-γ and Tc17/IFN-γ populations were markedly reduced in CD4-SOCS3KO, suggesting that SOCS3 promotes expansion of Th17/IFN-γ subset associated with development of severe uveitis. Thus, SOCS3 is a potential therapeutic target in uveitis and other auto-inflammatory diseases. PMID:24101549

SOCS3 deletion in T lymphocytes suppresses development of chronic ocular inflammation via upregulation of CTLA-4 and expansion of regulatory T cells.

PubMed

Yu, Cheng-Rong; Kim, Sung-Hye; Mahdi, Rashid M; Egwuagu, Charles E

2013-11-15

Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of the JAK/STAT pathway, and SOCS3 contributes to host immunity by regulating the intensity and duration of cytokine signals and inflammatory responses. Mice with Socs3 deletion in myeloid cells exhibit enhanced STAT3 signaling, expansion of Th1 and Th17 cells, and develop severe experimental autoimmune encephalomyelitis. Interestingly, development of the unique IL-17/IFN-γ double-producing (Th17/IFN-γ and Tc17/IFN-γ) subsets that exhibit strong cytotoxic activities and are associated with pathogenesis of several autoimmune diseases has recently been shown to depend on epigenetic suppression of SOCS3 expression, further suggesting involvement of SOCS3 in autoimmunity and tumor immunity. In this study, we generated mice with Socs3 deletion in the CD4 T cell compartment (CD4-SOCS3 knockout [KO]) to determine in vivo effects of the loss of Socs3 in the T cell-mediated autoimmune disease, experimental autoimmune uveitis (EAU). In contrast to the exacerbation of experimental autoimmune encephalomyelitis in myeloid-specific SOCS3-deleted mice, CD4-SOCS3KO mice were protected from acute and chronic uveitis. Protection from EAU correlated with enhanced expression of CTLA-4 and expansion of IL-10-producing regulatory T cells with augmented suppressive activities. We further show that SOCS3 interacts with CTLA-4 and negatively regulates CTLA-4 levels in T cells, providing a mechanistic explanation for the expansion of regulatory T cells in CD4-SOCS3 during EAU. Contrary to in vitro epigenetic studies, Th17/IFN-γ and Tc17/IFN-γ populations were markedly reduced in CD4-SOCS3KO, suggesting that SOCS3 promotes expansion of the Th17/IFN-γ subset associated with development of severe uveitis. Thus, SOCS3 is a potential therapeutic target in uveitis and other autoinflammatory diseases.

Attraction thresholds and sex discrimination of urinary odorants in male and female aromatase knockout (ArKO) mice.

PubMed

Pierman, Sylvie; Douhard, Quentin; Balthazart, Jacques; Baum, Michael J; Bakker, Julie

2006-01-01

We previously found that both male and female aromatase knockout (ArKO) mice, which cannot synthesize estrogens due to a targeted mutation of the aromatase gene, showed less investigation of volatile body odors from anesthetized conspecifics of both sexes in Y-maze tests. We now ask whether ArKO mice are in fact capable of discriminating between and/or responding to volatile odors. Using habituation/dishabituation tests, we found that gonadectomized ArKO and wild-type (WT) mice of both sexes, which were tested without any sex hormone replacement, reliably distinguished between undiluted volatile urinary odors of either adult males or estrous females versus deionized water as well as between these two urinary odors themselves. However, ArKO mice of both sexes were less motivated than WT controls to investigate same-sex odors when they were presented last in the sequence of stimuli. In a second experiment, we compared the ability of ArKO and WT mice to respond to decreasing concentrations of either male or female urinary odors. We found a clear-cut sex difference in urinary odor attraction thresholds among WT mice: WT males failed to respond to urine dilutions higher than 1:20 by volume, whereas WT females continued to respond to urine dilutions up to 1:80. Male ArKO mice resembled WT females in their ability to respond to lower concentrations of urinary odors, raising the possibility that the observed sex difference among WT mice in urine attraction thresholds results from the perinatal actions of estrogen in the male nervous system. Female ArKO mice failed to show significant dishabituation responses to two (1:20 and 1:80) dilutions of female urine, perhaps, again, because of a reduced motivation to investigate less salient, same-sex urinary odors. Previously observed deficits in the preference of ArKO male and female mice to approach volatile body odors from conspecifics of either sex cannot be attributed to an inability of ArKO subjects to discriminate these odors according to sex but instead may reflect a deficient motivation to approach same-sex odors, especially when their concentration is low.

Deletion of Glutamate Delta-1 Receptor in Mouse Leads to Enhanced Working Memory and Deficit in Fear Conditioning

PubMed Central

Yadav, Roopali; Hillman, Brandon G.; Gupta, Subhash C.; Suryavanshi, Pratyush; Bhatt, Jay M.; Pavuluri, Ratnamala; Stairs, Dustin J.; Dravid, Shashank M.

2013-01-01

Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system. PMID:23560106

The role of T1r3 and Trpm5 in carbohydrate-induced obesity in mice

PubMed Central

Glendinning, John I.; Gillman, Jennifer; Zamer, Haley; Margolskee, Robert F.; Sclafani, Anthony

2012-01-01

We examined the role of T1r3 and Trpm5 taste signaling proteins in carbohydrate-induced overeating and obesity. T1r3, encoded by Tas1r3, is part of the T1r2+T1r3 sugar taste receptor, while Trpm5 mediates signaling for G protein-coupled receptors in taste cells. It is known that C57BL/6 wild-type (WT) and Tas1r3 knock-out (KO) mice are attracted to the taste of Polycose (a glucose polymer), but not sucrose. In contrast, Trpm5 KO mice are not attracted to the taste of sucrose or Polycose. In Experiment 1, we maintained the WT, Tas1r3 KO and Trpm5 KO mice on one of three diets for 38 days: lab chow plus water (Control diet); chow, water and 34% Polycose solution (Polycose diet); or chow, water and 34% sucrose solution (Sucrose diet). The WT and Tas1r3 KO mice overconsumed the Polycose diet and became obese. The WT and Tas1r3 KO mice also overconsumed the Sucrose diet, but only the WT mice became obese. The Trpm5 KO mice, in contrast, showed little or no overeating on the Sucrose and Polycose diets, and gained slightly or significantly less weight than WT mice on these diets. In Experiment 2, we asked whether the Tas1r3 KO mice exhibited impaired weight gain on the Sucrose diet because it was insipid. To test this hypothesis, we maintained the WT and Tas1r3 KO mice on one of two diets for 38 days: chow, water and a dilute (1%) but highly palatable Intralipid emulsion (Control diet); or chow, water and a 34% sucrose + 1% Intralipid solution (Suc+IL diet). The WT and Tas1r3 KO mice both gained weight and became obese on the Suc+IL diet. Our results suggest that nutritive solutions must be highly palatable to cause carbohydrate-induced obesity in mice, and that palatability produces this effect in part by enhancing nutrient utilization. PMID:22683548

Metabotropic glutamate receptor subtype 7 has critical roles in regulation of the endocrine system and social behaviours.

PubMed

Masugi-Tokita, M; Yoshida, T; Kageyama, S; Kawata, M; Kawauchi, A

2018-03-01

Metabotropic glutamate receptor subtype 7 (mGluR7) is one of the group III mGluRs, which are negatively coupled to adenylate cyclase via Gi/Go proteins and localised to presynaptic active zones of the mammalian central nervous system. We previously reported that mGluR7 is essential for intermale aggression and amygdala-dependent fear learning. To elucidate the role of mGluR7 in the neuroendocrine system, we performed biochemical analyses and found a significant reduction of testosterone levels in mGluR7 knockout (KO) mice. Testosterone replacement restored intermale aggressive behaviour in castrated wild-type mice to the level of gonadally intact wild-type mice. However, given the same dosage of testosterone replacement, mGluR7 KO mice showed almost no aggressive behaviour. These results indicate that reduction of plasma testosterone is unrelated to the deficit in intermale aggression in mGluR7 KO mice. Social investigating behaviour of intact mGluR7 KO mice also differed from that of wild-type mice; e.g. the KO mice showing less frequent anogenital sniffing and more frequent grooming behaviour. Testosterone replacement increased anogenital sniffing and grooming behaviour in castrated mGluR7 KO mice, while the differences were still present between castrated wild-type mice and KO mice after both underwent testosterone replacement. These results imply that reduction of plasma testosterone may partially inhibit social investigating behaviours in intact mGluR7 KO mice. Furthermore, castrated mGluR7 KO mice have smaller seminal vesicles than those of castrated wild-type mice, although seminal vesicle weights were normal in intact mice. These observations suggest that, besides testicular testosterone, some other hormone levels may be dysregulated in mGluR7 KO mice, and indicate a critical role of mGluR7 in the endocrine system. Taken together, our findings demonstrate that mGluR7 is essential for the regulation of the endocrine system, in addition to innate behaviours such as intermale aggression and fear response. © 2018 British Society for Neuroendocrinology.

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

PubMed

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.

Endogenous nociceptin modulates diet preference independent of motivation and reward.

PubMed

Koizumi, Miwako; Cagniard, Barbara; Murphy, Niall P

2009-04-20

Previous studies show that the opioid peptide nociceptin stimulates food intake. Here, we studied nociceptin receptor knockout (NOP KO) mice in various behavioral paradigms designed to differentiate psychological and physiological loci at which endogenous nociceptin might control feeding. When presented a choice under food restriction, NOP KO mice displayed reduced preference for high sucrose diet, but lower intake of high fat diet under no-choice conditions. These responses were absent under ad libitum feeding conditions. Conditioned place preference to high fat diet under food-deprived conditions was unaltered in NOP KO mice, suggesting no difference in reward responses. Furthermore, operant food self-administration under a variety of conditions showed no genotype-dependent differences, suggesting no differences in the motivational properties of food. Taste reactivity to sucrose was unchanged in NOP KO mice, though NOP KO mice had altered aversive reactions to quinine solutions under ad libitum feeding, suggesting minor differences in the affective impact of palatable and unpalatable tastants. Although NOP KO mice re-fed following food-deprivation showed normal increases in plasma glucose and insulin, multidimensional scaling analysis showed that the relationship between these measures, body weight and plasma leptin was substantially disrupted in NOP KO, particularly in fasted mice. Additionally, the typical positive relationship between body weight and plasma leptin was considerably weaker in NOP KO mice. Together, these findings suggest that endogenous nociceptin differentially modulates diet preference depending on macronutrient content and homeostatic state, independently of the motivating, rewarding or orosensory properties of food, but may involve metabolic or postingestive processes.

New insight into the role of MMP14 in metabolic balance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mori, Hidetoshi; Bhat, Ramray; Bruni-Cardoso, Alexandre

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout of Mmp14 (KO) in mice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organ model for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into anmore » unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO rudiments were reduced, and the circulating levels of triglycerides and glucose were lower. Analysis of the ultrastructure of mammary glands imaged by electron microscopy revealed a significant increase in autophagy signatures in KO mice. Finally, Mmp14 silenced mammary epithelial cells displayed enhanced autophagy. Applied to a systemic level, these findings indicate that MMP14 is a crucial regulator of tissue homeostasis. If operative on a systemic level, these findings could explain how Mmp14KO litter fail to thrive due to disorder in metabolism.« less

Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas

PubMed Central

Okada, Yuka; Shirai, Kumi; Miyajima, Masayasu; Reinach, Peter S.; Yamanaka, Osamu; Sumioka, Takayoshi; Kokado, Masahide; Tomoyose, Katsuo; Saika, Shizuya

2016-01-01

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor β, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice. PMID:28030558

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

PubMed Central

Prossnitz, Eric R.; Hathaway, Helen J.

2015-01-01

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

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

PubMed

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.

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium*

PubMed Central

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-01-01

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg2+-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6–8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. PMID:26504086

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium.

PubMed

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-12-11

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg(2+)-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6-8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

New insight into the role of MMP14 in metabolic balance

DOE PAGES

Mori, Hidetoshi; Bhat, Ramray; Bruni-Cardoso, Alexandre; ...

2016-07-13

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout of Mmp14 (KO) in mice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organ model for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into anmore » unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO rudiments were reduced, and the circulating levels of triglycerides and glucose were lower. Analysis of the ultrastructure of mammary glands imaged by electron microscopy revealed a significant increase in autophagy signatures in KO mice. Finally, Mmp14 silenced mammary epithelial cells displayed enhanced autophagy. Applied to a systemic level, these findings indicate that MMP14 is a crucial regulator of tissue homeostasis. If operative on a systemic level, these findings could explain how Mmp14KO litter fail to thrive due to disorder in metabolism.« less

Glutathione-deficient Plasmodium berghei parasites exhibit growth delay and nuclear DNA damage.

PubMed

Padín-Irizarry, Vivian; Colón-Lorenzo, Emilee E; Vega-Rodríguez, Joel; Castro, María Del R; González-Méndez, Ricardo; Ayala-Peña, Sylvette; Serrano, Adelfa E

2016-06-01

Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

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. PMID:27649573

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

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress.

PubMed

Yagishita, Yoko; Uruno, Akira; Fukutomi, Toshiaki; Saito, Ritsumi; Saigusa, Daisuke; Pi, Jingbo; Fukamizu, Akiyoshi; Sugiyama, Fumihiro; Takahashi, Satoru; Yamamoto, Masayuki

2017-02-21

The relationship between loss of hypothalamic function and onset of diabetes mellitus remains elusive. Therefore, we generated a targeted oxidative-stress murine model utilizing conditional knockout (KO) of selenocysteine-tRNA (Trsp) using rat-insulin-promoter-driven-Cre (RIP-Cre). These Trsp-KO (Trsp RIP KO) mice exhibit deletion of Trsp in both hypothalamic cells and pancreatic β cells, leading to increased hypothalamic oxidative stress and severe insulin resistance. Leptin signals are suppressed, and numbers of proopiomelanocortin-positive neurons in the hypothalamus are decreased. In contrast, Trsp-KO mice (Trsp Ins1 KO) expressing Cre specifically in pancreatic β cells, but not in the hypothalamus, do not display insulin and leptin resistance, demonstrating a critical role of the hypothalamus in the onset of diabetes mellitus. Nrf2 (NF-E2-related factor 2) regulates antioxidant gene expression. Increased Nrf2 signaling suppresses hypothalamic oxidative stress and improves insulin and leptin resistance in Trsp RIP KO mice. Thus, Nrf2 harbors the potential to prevent the onset of diabetic mellitus by reducing hypothalamic oxidative damage. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice.

PubMed

Kawamata, Masaki; Yoshida, Masahide; Sugimoto, Yukihiko; Kimura, Tadashi; Tonomura, Yutaka; Takayanagi, Yuki; Yanagisawa, Teruyuki; Nishimori, Katsuhiko

2008-02-13

The dramatic increase of oxytocin (OT) receptor (OTR) in the myometrium as well as circulating progesterone withdrawal has been thought to be the most important factor in the induction and accomplishment of parturition since delivery fails in prostaglandin F2alpha receptor (FP) knockout (FP KO) mice. The expression levels of OTR mRNA/protein were not dramatically increased in the near-term uteri of FP KO mice. However, OT-induced myometrial contractions and the concentration-response curves in FP KO in vitro were almost similar to those in wild-type (WT) mice. OT-infusion (0.3 U/day) enabled FP KO mice to experience successful delivery, and furthermore the duration until the onset was hastened by a higher dose of OT (3 U/day). The plasma progesterone levels of FP KO females were maintained at high levels, but decreased during labor by OT-infusion (3 U/day). These results suggest that OT has potentials to induce strong myometrial contractions in uterus with low expression levels of OTR and luteolysis in ovary, which enabled FP KO females to undergo successful delivery.

Improved Oxygen Sources for Breathing Apparatus

NASA Technical Reports Server (NTRS)

Wood, P. C.; Wydeven, T.

1983-01-01

Research is described which is directed toward the preparation of chemical oxygen sources which exhibited improved O2 storage and reaction characteristics when compared to potassium superoxide (KO2). The initial focus of the research was the preparation of calcium superoxide (Ca(O2)2) by the disproportionation of calcium peroxide diperoxyhydrate. the Ca(O2)2 was characterized by chemical, thermal, and x ray analyses. Several methods for scaling up the Ca(O2)2 syntheis process were studied. The reactivity of Ca(O2)2 toward humidified carbon dioxide (CO2) was evaluated and was compared to that of KO2 under flow test conditions approximating those existing in portable breathing apparatus. The reactivities of mixtures of KO2 and Ca(O2)2 or lithium peroxide towards humidified CO2 were also studied. Finally, an analysis of two commercial, KO2-based, self contained self rescuers was conducted to determine the potential weight and volume savings which would be possible if Ca(O2)2 or a mixture of KO2 and Ca(O2)2 were used as a replacement for KO2.

Diabetes accelerates retinal ganglion cell dysfunction in mice lacking sigma receptor 1

PubMed Central

Ha, Yonju; Saul, Alan; Tawfik, Amany; Zorrilla, Eric P.; Ganapathy, Vadivel

2012-01-01

Purpose Sigma receptor 1 (σR1) is a non-opioid transmembrane protein that may act as a molecular chaperone at the endoplasmic reticulum–mitochondrial membrane. Ligands for σR1, such as (+)-pentazocine [(+)-PTZ], confer marked retinal neuroprotection in vivo and in vitro. Recently we analyzed the retinal phenotype of mice lacking σR1 (σR1 KO) and observed normal retinal morphology and function in young mice (5–30 weeks) but diminished negative scotopic threshold responses (nSTRs), retinal ganglion cell (RGC) loss, and disruption of optic nerve axons consistent with inner retinal dysfunction by 1 year. These data led us to test the hypothesis that σR1 may be critical in forestalling chronic retinal stress; diabetes was used as the model of chronic stress. Methods To determine whether σR1 is required for (+)-PTZ neuroprotective effects, primary RGCs isolated from wild-type (WT) and σR1 KO mice were exposed to xanthine–xanthine oxidase (10 µM:2 mU/ml) to induce oxidative stress in the presence or absence of (+)-PTZ. Cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. To assess effects of chronic stress on RGC function, diabetes was induced in 3-week C57BL/6 (WT) and σR1 KO mice, using streptozotocin to yield four groups: WT nondiabetic (WT non-DB), WT diabetic (WT-DB), σR1 KO non-DB, and σR1 KO-DB. After 12 weeks of diabetes, when mice were 15-weeks old, intraocular pressure (IOP) was recorded, electrophysiologic testing was performed (including detection of nSTRs), and the number of RGCs was counted in retinal histological sections. Results In vitro studies showed that (+)-PTZ could not prevent oxidative stress-induced death of RGCs harvested from σR1 KO mice but afforded robust protection against death of RGCs harvested from WT mice. In the studies of chronic stress induced by diabetes, the IOP measured in the four mouse groups was within the normal range; however, there was a significant increase in the IOP of σR1 KO-DB mice (16±0.5 mmHg) compared to the other groups tested (σR1 KO non-DB, WT non-DB, WT-DB: ~12±0.6 mmHg). Regarding electrophysiologic testing, the nSTRs of σR1 KO non-DB mice were similar to WT non-DB mice at 15 weeks; however, they were significantly lower in σR1 KO-DB mice (5±1 µV) compared to the other groups, including, notably, σR1 KO-nonDB (12±2 µV). As expected, the number of RGCs in σR1 KO non-DB mice was similar to WT non-DB mice at 15 weeks, but under chronic stress of diabetes there were fewer RGCs in retinas of σR1 KO-DB mice. Conclusions This is the first report showing unequivocally that the neuroprotective effects of (+)-PTZ require σR1. σR1 KO mice show normal retinal structure and function at young ages; however, when subjected to the chronic stress of diabetes, there is an acceleration of retinal functional deficits in σR1 KO mice such that ganglion cell dysfunction is observed at a much earlier age than nondiabetic σR1 KO mice. The data support the hypothesis that σR1 plays a key role in modulating retinal stress and may be an important target for retinal disease. PMID:23233788

Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P).

PubMed

Massaguer, A; Engel, P; Pérez-del-Pulgar, S; Bosch, J; Pizcueta, P

2000-08-01

P-selectin (CD62P) is an adhesion molecule expressed on the activated endothelium and activated platelets that is involved in the initial attachment of leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodies (mAbs) and P-selectin-deficient mice have shown that P-selectin is a potential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology. The aim of this study was to generate a novel panel of anti-P-selectin mAbs against the conserved epitopes present in several animal species. To produce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recognize human P-selectin were obtained. Individual mAbs that bound to human, rat, mouse, rabbit and pig activated platelets were characterized by flow-cytometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P-sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL60 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MAb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELISA assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure soluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are unique since they recognize common epitopes conserved during mammalian evolution and they may be useful for studying P-selectin function in inflammatory models in various species.

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

PubMed

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.

TASK-3 knockout mice exhibit exaggerated nocturnal activity, impairments in cognitive functions, and reduced sensitivity to inhalation anesthetics.

PubMed

Linden, Anni-Maija; Sandu, Cristina; Aller, M Isabel; Vekovischeva, Olga Y; Rosenberg, Per H; Wisden, William; Korpi, Esa R

2007-12-01

The TASK-3 channel is an acid-sensitive two-pore-domain K+ channel, widely expressed in the brain and probably involved in regulating numerous neuronal populations. Here, we characterized the behavioral and pharmacological phenotypes of TASK-3 knockout (KO) mice. Circadian locomotor activity measurements revealed that the nocturnal activity of the TASK-3 KO mice was increased by 38% (P < 0.01) compared with wild-type littermate controls, light phase activity being similar. Although TASK-3 channels are abundant in cerebellar granule cells, the KO mice performed as well as the wild-type mice in walking on a rotating rod or along a 1.2-cm-diameter beam. However, they fell more frequently from a narrower 0.8-cm beam. The KO mice showed impaired working memory in the spontaneous alternation task, with the alternation percentage being 62 +/- 3% for the wild-type mice and 48 +/- 4% (P < 0.05) for the KO mice. Likewise, during training for the Morris water-maze spatial memory task, the KO mice were slower to find the hidden platform, and in the probe trial, the female KO mice visited fewer times the platform quadrant than the male KO and wild-type mice. In pharmacological tests, the TASK-3 KO mice showed reduced sensitivity to the inhalation anesthetic halothane and the cannabinoid receptor agonist WIN55212-2 mesylate [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] but unaltered responses to the alpha2 adrenoceptor agonist dexmedetomidine, the i.v. anesthetic propofol, the opioid receptor agonist morphine, and the local anesthetic lidocaine. Overall, our results suggest important contributions of TASK-3 channels in the neuronal circuits regulating circadian rhythms, cognitive functions, and mediating specific pharmacological effects.

Excessive Astrocyte-Derived Neurotrophin-3 Contributes to the Abnormal Neuronal Dendritic Development in a Mouse Model of Fragile X Syndrome

PubMed Central

Guo, Yan-yan; Liu, Shui-bing; Wu, Yu-mei; Li, Xiao-qiang; Zhao, Ming-gao

2012-01-01

Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO) mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM) from KO astrocytes inhibited proper dendritic growth of both wild-type (WT) and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3) in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were normal. FMRP has multiple RNA–binding motifs and is involved in translational regulation. RNA–binding protein immunoprecipitation (RIP) showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs). Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS. PMID:23300470

Clozapine Reverses Phencyclidine-Induced Desynchronization of Prefrontal Cortex through a 5-HT1A Receptor-Dependent Mechanism

PubMed Central

Kargieman, Lucila; Riga, Maurizio S; Artigas, Francesc; Celada, Pau

2012-01-01

The non-competitive NMDA receptor (NMDA-R) antagonist phencyclidine (PCP)—used as a pharmacological model of schizophrenia—disrupts prefrontal cortex (PFC) activity. PCP markedly increased the discharge rate of pyramidal neurons and reduced slow cortical oscillations (SCO; 0.15–4 Hz) in rat PFC. Both effects were reversed by classical (haloperidol) and atypical (clozapine) antipsychotic drugs. Here we extended these observations to mice brain and examined the potential involvement of 5-HT2A and 5-HT1A receptors (5-HT2AR and 5-HT1AR, respectively) in the reversal by clozapine of PCP actions. Clozapine shows high in vitro affinity for 5-HT2AR and behaves as partial agonist in vivo at 5-HT1AR. We used wild-type (WT) mice and 5-HT1AR and 5-HT2AR knockout mice of the same background (C57BL/6) (KO-1A and KO-2A, respectively). Local field potentials (LFPs) were recorded in the PFC of WT, KO-1A, and KO-2A mice. PCP (10 mg/kg, intraperitoneally) reduced SCO equally in WT, KO-2A, and KO-1A mice (58±4%, 42±7%, and 63±7% of pre-drug values, n=23, 13, 11, respectively; p<0.0003). Clozapine (0.5 mg/kg, intraperitoneally) significantly reversed PCP effect in WT and KO-2A mice, but not in KO-1A mice nor in WT mice pretreated with the selective 5-HT1AR antagonist WAY-100635.The PCP-induced disorganization of PFC activity does not appear to depend on serotonergic function. However, the lack of effect of clozapine in KO-1A mice and the prevention by WAY-100635 indicates that its therapeutic action involves 5-HT1AR activation without the need to block 5-HT2AR, as observed with clozapine-induced cortical dopamine release. PMID:22012474

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishizaka, Masanori; Gohda, Tomohito, E-mail: goda@juntendo.ac.jp; Takagi, Miyuki

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, althoughmore » 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.« less

Adipose tissue (P)RR regulates insulin sensitivity, fat mass and body weight.

PubMed

Shamansurova, Zulaykho; Tan, Paul; Ahmed, Basma; Pepin, Emilie; Seda, Ondrej; Lavoie, Julie L

2016-10-01

We previously demonstrated that the handle-region peptide, a prorenin/renin receptor [(P)RR] blocker, reduces body weight and fat mass and may improve insulin sensitivity in high-fat fed mice. We hypothesized that knocking out the adipose tissue (P)RR gene would prevent weight gain and insulin resistance. An adipose tissue-specific (P)RR knockout (KO) mouse was created by Cre-loxP technology using AP2-Cre recombinase mice. Because the (P)RR gene is located on the X chromosome, hemizygous males were complete KO and had a more pronounced phenotype on a normal diet (ND) diet compared to heterozygous KO females. Therefore, we challenged the female mice with a high-fat diet (HFD) to uncover certain phenotypes. Mice were maintained on either diet for 9 weeks. KO mice had lower body weights compared to wild-types (WT). Only hemizygous male KO mice presented with lower total fat mass, higher total lean mass as well as smaller adipocytes compared to WT mice. Although food intake was similar between genotypes, locomotor activity during the active period was increased in both male and female KO mice. Interestingly, only male KO mice had increased O2 consumption and CO2 production during the entire 24-hour period, suggesting an increased basal metabolic rate. Although glycemia during a glucose tolerance test was similar, KO males as well as HFD-fed females had lower plasma insulin and C-peptide levels compared to WT mice, suggesting improved insulin sensitivity. Remarkably, all KO animals exhibited higher circulating adiponectin levels, suggesting that this phenotype can occur even in the absence of a significant reduction in adipose tissue weight, as observed in females and, thus, may be a specific effect related to the (P)RR. (P)RR may be an important therapeutic target for the treatment of obesity and its associated complications such as type 2 diabetes.

Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

PubMed

Zhang, Min; Mongue-Din, Heloise; Martin, Daniel; Catibog, Norman; Smyrnias, Ioannis; Zhang, Xiaohong; Yu, Bin; Wang, Minshu; Brandes, Ralf P; Schröder, Katrin; Shah, Ajay M

2018-03-01

NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. © The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.

Functional β-Adrenoceptors Are Important for Early Muscle Regeneration in Mice through Effects on Myoblast Proliferation and Differentiation

PubMed Central

Church, Jarrod E.; Trieu, Jennifer; Sheorey, Radhika; Chee, Annabel Y. -M.; Naim, Timur; Baum, Dale M.; Ryall, James G.; Gregorevic, Paul; Lynch, Gordon S.

2014-01-01

Muscles can be injured in different ways and the trauma and subsequent loss of function and physical capacity can impact significantly on the lives of patients through physical impairments and compromised quality of life. The relative success of muscle repair after injury will largely determine the extent of functional recovery. Unfortunately, regenerative processes are often slow and incomplete, and so developing novel strategies to enhance muscle regeneration is important. While the capacity to enhance muscle repair by stimulating β2-adrenoceptors (β-ARs) using β2-AR agonists (β2-agonists) has been demonstrated previously, the exact role β-ARs play in regulating the regenerative process remains unclear. To investigate β-AR-mediated signaling in muscle regeneration after myotoxic damage, we examined the regenerative capacity of tibialis anterior and extensor digitorum longus muscles from mice lacking either β1-AR (β1-KO) and/or β2-ARs (β2-KO), testing the hypothesis that muscles from mice lacking the β2-AR would exhibit impaired functional regeneration after damage compared with muscles from β1-KO or β1/β2-AR null (β1/β2-KO) KO mice. At 7 days post-injury, regenerating muscles from β1/β2-KO mice produced less force than those of controls but muscles from β1-KO or β2-KO mice did not exhibit any delay in functional restoration. Compared with controls, β1/β2-KO mice exhibited an enhanced inflammatory response to injury, which delayed early muscle regeneration, but an enhanced myoblast proliferation later during regeneration ensured a similar functional recovery (to controls) by 14 days post-injury. This apparent redundancy in the β-AR signaling pathway was unexpected and may have important implications for manipulating β-AR signaling to improve the rate, extent and efficacy of muscle regeneration to enhance functional recovery after injury. PMID:25000590

Deletion of AMPKα1 attenuates the anticontractile effect of perivascular adipose tissue (PVAT) and reduces adiponectin release.

PubMed

Almabrouk, Tarek A M; Ugusman, Azizah B; Katwan, Omar J; Salt, Ian P; Kennedy, Simon

2017-10-01

Perivascular adipose tissue (PVAT) surrounds most blood vessels and secretes numerous active substances, including adiponectin, which produce a net anticontractile effect in healthy individuals. AMPK is a key mediator of cellular energy balance and may mediate the vascular effects of adiponectin. In this study, we investigated the role of AMPK within PVAT in mediating the anticontractile effect of PVAT. Endothelium-denuded aortic rings from wild-type (WT; Sv129) and α 1 AMPK knockout (KO) mice were mounted on a wire myograph. Dose-response curves to the AMPK-independent vasodilator cromakalim were studied in vessels with and without PVAT, and effect of pre-incubation with conditioned media and adiponectin on relaxation was also studied. The effect of AMPKα1 KO on the secretory profile of PVAT was assessed by elisa. Thoracic aortic PVAT from KO mice was morphologically indistinct from that of WT and primarily composed of brown adipose tissue. PVAT augmented relaxation to cromakalim in WT but not KO aortic rings. Addition of WT PVAT augmented relaxation in KO aortic rings but KO PVAT had no effect in WT rings. PVAT from KO mice secreted significantly less adiponectin and addition of adiponectin to either KO or WT aortic rings without PVAT augmented relaxation to cromakalim. An adiponectin blocking peptide significantly attenuated relaxation in WT rings with PVAT but not in KO rings. AMPKα1 has a critical role in maintaining the anticontractile actions of PVAT; an effect independent of the endothelium but likely mediated through altered adiponectin secretion or sensitivity. This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc. © 2016 The British Pharmacological Society.

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

PubMed

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

2015-05-01

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

p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armesilla-Diaz, Alejandro, E-mail: aarmesilla@cib.csic.es; Elvira, Gema; Silva, Augusto

2009-12-10

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC, from both human and murine origin, has been reported in many studies. MSC transformation depends on the culture conditions, the origin of the cells and the time on culture; however, the precise biological characteristics involved in this process have not been fully defined yet. In this study, we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate, amore » shorter doubling time and also morphologic and phenotypic changes, as compared to MSC derived from wild-type animals. Furthermore, the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition, not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover, genomic instability, changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition, the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC.« less

Hypothyroidism Compromises Hypothalamic Leptin Signaling in Mice

PubMed Central

Groba, Claudia; Mayerl, Steffen; van Mullem, Alies A.; Visser, Theo J.; Darras, Veerle M.; Habenicht, Andreas J.

2013-01-01

The impact of thyroid hormone (TH) on metabolism and energy expenditure is well established, but the role of TH in regulating nutritional sensing, particularly in the central nervous system, is only poorly defined. Here, we studied the consequences of hypothyroidism on leptin production as well as leptin sensing in congenital hypothyroid TRH receptor 1 knockout (Trhr1 ko) mice and euthyroid control animals. Hypothyroid mice exhibited decreased circulating leptin levels due to a decrease in fat mass and reduced leptin expression in white adipose tissue. In neurons of the hypothalamic arcuate nucleus, hypothyroid mice showed increased leptin receptor Ob-R expression and decreased suppressor of cytokine signaling 3 transcript levels. In order to monitor putative changes in central leptin sensing, we generated hypothyroid and leptin-deficient animals by crossing hypothyroid Trhr1 ko mice with the leptin-deficient ob/ob mice. Hypothyroid Trhr1/ob double knockout mice showed a blunted response to leptin treatment with respect to body weight and food intake and exhibited a decreased activation of phospho-signal transducer and activator of transcription 3 as well as a up-regulation of suppressor of cytokine signaling 3 upon leptin treatment, particularly in the arcuate nucleus. These data indicate alterations in the intracellular processing of the leptin signal under hypothyroid conditions and thereby unravel a novel mode of action by which TH affects energy metabolism. PMID:23518925

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

PubMed Central

Filippini, Alice; Bonini, Daniela; Lacoux, Caroline; Zingariello, Maria; Sancillo, Laura; Bosisio, Daniela; Salvi, Valentina; Mingardi, Jessica; La Via, Luca; Zalfa, Francesca; Bagni, Claudia

2017-01-01

ABSTRACT The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes. PMID:28640668

Selective Biological Responses of Phagocytes and Lungs to Purified Histones.

PubMed

Fattahi, Fatemeh; Grailer, Jamison J; Lu, Hope; Dick, Rachel S; Parlett, Michella; Zetoune, Firas S; Nuñez, Gabriel; Ward, Peter A

2017-01-01

Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1β was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4. © 2017 S. Karger AG, Basel.

The skeletal structure of insulin-like growth factor I-deficient mice

NASA Technical Reports Server (NTRS)

Bikle, D.; Majumdar, S.; Laib, A.; Powell-Braxton, L.; Rosen, C.; Beamer, W.; Nauman, E.; Leary, C.; Halloran, B.

2001-01-01

The importance of insulin-like growth factor I (IGF-I) for growth is well established. However, the lack of IGF-I on the skeleton has not been examined thoroughly. Therefore, we analyzed the structural properties of bone from mice rendered IGF-I deficient by homologous recombination (knockout [k/o]) using histomorphometry, peripheral quantitative computerized tomography (pQCT), and microcomputerized tomography (muCT). The k/o mice were 24% the size of their wild-type littermates at the time of study (4 months). The k/o tibias were 28% and L1 vertebrae were 26% the size of wild-type bones. Bone formation rates (BFR) of k/o tibias were 27% that of the wild-type littermates. The k/o bones responded normally to growth hormone (GH; 1.7-fold increase) and supranormally to IGF-I (5.2-fold increase) with respect to BFR. Cortical thickness of the proximal tibia was reduced 17% in the k/o mouse. However, trabecular bone volume (bone volume/total volume [BV/TV]) was increased 23% (male mice) and 88% (female mice) in the k/o mice compared with wild-type controls as a result of increased connectivity, increased number, and decreased spacing of the trabeculae. These changes were either less or not found in L1. Thus, lack of IGF-I leads to the development of a bone structure, which, although smaller, appears more compact.

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

PubMed

Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

PubMed

Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

2016-10-01

Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth. Copyright © 2016 Elsevier B.V. All rights reserved.

Zinc transporter 3 is involved in learned fear and extinction, but not in innate fear.

PubMed

Martel, Guillaume; Hevi, Charles; Friebely, Olivia; Baybutt, Trevor; Shumyatsky, Gleb P

2010-11-01

Synaptically released Zn²+ is a potential modulator of neurotransmission and synaptic plasticity in fear-conditioning pathways. Zinc transporter 3 (ZnT3) knock-out (KO) mice are well suited to test the role of zinc in learned fear, because ZnT3 is colocalized with synaptic zinc, responsible for its transport to synaptic vesicles, highly enriched in the amygdala-associated neural circuitry, and ZnT3 KO mice lack Zn²+ in synaptic vesicles. However, earlier work reported no deficiency in fear memory in ZnT3 KO mice, which is surprising based on the effects of Zn²+ on amygdala synaptic plasticity. We therefore reexamined ZnT3 KO mice in various tasks for learned and innate fear. The mutants were deficient in a weak fear-conditioning protocol using single tone-shock pairing but showed normal memory when a stronger, five-pairing protocol was used. ZnT3 KO mice were deficient in memory when a tone was presented as complex auditory information in a discontinuous fashion. Moreover, ZnT3 KO mice showed abnormality in trace fear conditioning and in fear extinction. By contrast, ZnT3 KO mice had normal anxiety. Thus, ZnT3 is involved in associative fear memory and extinction, but not in innate fear, consistent with the role of synaptic zinc in amygdala synaptic plasticity.

Stem cell antigen-1 in skeletal muscle function.

PubMed

Bernstein, Harold S; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-08-15

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.

CREB binding protein is required for both short-term and long-term memory formation.

PubMed

Chen, Guiquan; Zou, Xiaoyan; Watanabe, Hirotaka; van Deursen, Jan M; Shen, Jie

2010-09-29

CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated conditional knock-out (cKO) mice in which CBP is completely inactivated in excitatory neurons of the postnatal forebrain. Histological analysis revealed normal neuronal morphology and absence of age-dependent neuronal degeneration in the CBP cKO cerebral cortex. CBP cKO mice exhibited robust impairment in the formation of spatial, associative, and object-recognition memory. In addition to impaired long-term memory, CBP cKO mice also displayed deficits in short-term associative and object-recognition memory. Administration of a histone deacetylase inhibitor, trichostatin A, rescued the reduction of acetylated histones in the CBP cKO cortex but failed to rescue either short- or long-term memory deficits, suggesting that the memory impairment may not be caused by general reduction of histone acetyltransferase activity in CBP cKO mice. Further microarray and Western analysis showed decreased expression of calcium-calmodulin-dependent kinase isoforms and NMDA and AMPA receptor subunits in the cerebral cortex of CBP cKO mice. Collectively, these findings suggest a crucial role for CBP in the formation of both short- and long-term memory.

Role of naturally occurring CD4+ CD25+ regulatory T cells in experimental atherosclerosis.

PubMed

Mor, Adi; Planer, David; Luboshits, Galia; Afek, Arnon; Metzger, Shula; Chajek-Shaul, Tova; Keren, Gad; George, Jacob

2007-04-01

Naturally occurring CD4+ CD25+ regulatory T cells (Tregs) exert suppressive effects on effector CD4 cells and downregulate experimental autoimmune disorders. We investigated the importance and potential role of Tregs in murine atherogenesis. Tregs were investigated comparatively between aged and young apolipoprotein E-knockout (ApoE-KO) mice and age-matched C57BL/6 littermates. The effect of oxidized LDL (oxLDL) was tested on the functional suppressive properties of Tregs from ApoE-KO and C57BL/6 mice. Tregs, CD4+ CD25- cells, and saline were infused into ApoE-KO mice to study their effects on atherogenesis. Treg numbers were reduced in atherosclerotic compared with nonatherosclerotic ApoE-KO mice. The functional suppressive properties of Tregs from ApoE-KO mice were compromised in comparison with those from their C57BL/6 littermates. Thus, oxLDL attenuated the suppressive properties of Tregs from C57BL/6 mice and more so in ApoE-KO mice. Transfer of Tregs from age-matched ApoE-KO mice resulted in significant attenuation of atherosclerosis compared with that after delivery of CD4+ CD25+/- T cells or phosphate-buffered saline. CD4+ CD25+ Tregs may play a protective role in the progression of atherosclerosis and could be considered a therapeutic tool if results from human studies can solidify observations in murine models.

Adenylyl cyclase-5 in the dorsal striatum function as a molecular switch for the generation of behavioral preferences for cue-directed food choices.

PubMed

Kim, Hannah; Kim, Tae-Kyung; Kim, Ji-Eun; Park, Jin-Young; Lee, Yunjin; Kang, Minkyung; Kim, Kyoung-Shim; Han, Pyung-Lim

2014-11-07

Behavioral choices in habits and innate behaviors occur automatically in the absence of conscious selection. These behaviors are not easily modified by learning. Similar types of behaviors also occur in various mental illnesses including drug addiction, obsessive-compulsive disorder, schizophrenia, and autism. However, underlying mechanisms are not clearly understood. In the present study, we investigated the molecular mechanisms regulating unconditioned preferred behaviors in food-choices. Mice lacking adenylyl cyclase-5 (AC5 KO mice), which is preferentially expressed in the dorsal striatum, consumed food pellets nearly one after another in cages. AC5 KO mice showed aversive behaviors to bitter tasting quinine, but they compulsively chose quinine-containing AC5 KO-pellets over fresh pellets. The unusual food-choice behaviors in AC5 KO mice were due to the gain of behavioral preferences for food pellets containing an olfactory cue, which wild-type mice normally ignored. Such food-choice behaviors in AC5 KO mice disappeared when whiskers were trimmed. Conversely, whisker trimming in wildtype mice induced behavioral preferences for AC5 KO food pellets, indicating that preferred food-choices were not learned through prior experience. Both AC5 KO mice and wildtype mice with trimmed whiskers had increased glutamatergic input from the barrel cortex into the dorsal striatum, resulting in an increase in the mGluR1-dependent signaling cascade. The siRNA-mediated inhibition of mGluR1 in the dorsal striatum in AC5 KO mice and wildtype mice with trimmed whiskers abolished preferred choices for AC5 KO food pellets, whereas siRNA-mediated inhibition of mGluR3 glutamate receptors in the dorsal striatum in wildtype mice induced behavioral preferences for AC5 KO food pellets, thus mimicking AC5 KO phenotypes. Our results show that the gain and loss of behavioral preferences for a specific cue-directed option were regulated by specific cellular factors in the dorsal striatum, such that the preferred food choices were switched on when either the mGluR3-AC5 pathway was inactive or the mGluR1 pathway was active, whereas the preferred food-choices were switched off when mGluR1 or its downstream pathway was suppressed. These results identify the AC5 and mGluR system in the dorsal striatum as molecular on/off switches to direct decisions on behavioral preferences for cue-oriented options.

Inquiries into the Biological Significance of Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8 Through Investigations of TARP γ-8 Null Mice§.

PubMed

Gleason, Scott D; Kato, Akihiko; Bui, Hai H; Thompson, Linda K; Valli, Sabrina N; Stutz, Patrick V; Kuo, Ming-Shang; Falcone, Julie F; Anderson, Wesley H; Li, Xia; Witkin, Jeffrey M

2015-01-01

Transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory protein (TARP) γ-8 is an auxiliary protein associated with some AMPA receptors. Most strikingly, AMPA receptors associated with this TARP have a relatively high localization in the hippocampus. TARP γ-8 also modifies the pharmacology and trafficking of AMPA receptors. However, to date there is little understanding of the biological significance of this auxiliary protein. In the present set of studies we provide a characterization of the differential pharmacology and behavioral consequences of deletion of TARP γ-8 by comparing the wild type (WT) and γ-8 -/- (knock-out, KO) mouse. KO mice were mildly hyperactive in a locomotor arena but not in other environments compared to WT mice. Additionally, the KO mice demonstrated enhanced locomotor stimulatory effects of both d-amphetamine and phencyclidine. Marble-burying and digging behaviors were dramatically reduced in KO mice. In another assay that can detect anxiety-like phenotypes, the elevated plus maze, no differences were observed in overall movement or open arm entries. In the forced-swim assay, KO mice displayed decreases in immobility time like the antidepressant imipramine and the AMPA receptor potentiator, LY392098. In KO mice, the antidepressant-like effects of LY392098 were prevented whereas the effects of imipramine were unaffected. Convulsions were induced by pentylenetetrazole, N-methyl-D-aspartate, and by kainic acid. However, in KO mice, kainic acid produced less tonic convulsions and lethality. KO mice had reduced levels of norepinephrine in hippocampus and cerebellum but not in hypothalamus or prefrontal cortex, decreased levels of cAMP in hippocampus, and increased levels of acetylcholine in the hypothalamus and prefrontal cortex. KO mice displayed decreased turnover of dopamine and increased histamine turnover in multiple brain areas In contrast, serotonin and its metabolites were not significantly affected by deletion of the γ-8 protein. Of a large panel of plasma lipids, only two monoacylglycerols (1OG and 2OG) were marginally but nonsignificantly altered in WT vs KO mice. Overall, the data suggest genetic inactivation of this specific population of AMPA receptors results in modest changes in behavior characterized by a mild hyperactivity which is condition dependent and a marked reduction in digging and burying behaviors. Despite deletion of TARP γ-8, chemoconvulsants were still active. Consistent with their predicted pharmacological actions, the convulsant effects of kainate and the antidepressant-like effects of an AMPA receptor potentiator (both acting upon AMPA receptors) were reduced or absent in KO mice.

NCKX3 was compensated by calcium transporting genes and bone resorption in a NCKX3 KO mouse model.

PubMed

Yang, Hyun; Ahn, Changhwan; Shin, Eun-Kyeong; Lee, Ji-Sun; An, Beum-Soo; Jeung, Eui-Bae

2017-10-15

Gene knockout is the most powerful tool for determination of gene function or permanent modification of the phenotypic characteristics of an animal. Existing methods for gene disruption are limited by their efficiency, time required for completion and potential for confounding off-target effects. In this study, a rapid single-step approach to knockout of a targeted gene in mice using zinc-finger nucleases (ZFNs) was demonstrated for generation of mutant (knockout; KO) alleles. Specifically, ZFNs to target the sodium/calcium/potassium exchanger3 (NCKX3) gene in C57bl/6j were designed using the concept of this approach. NCKX3 KO mice were generated and the phenotypic characterization and molecular regulation of active calcium transporting genes was assessed when mice were fed different calcium diets during growth. General phenotypes such as body weight and plasma ion level showed no distinct abnormalities. Thus, the potassium/sodium/calcium exchanger of NCKX3 KO mice proceeded normally in this study. As a result, the compensatory molecular regulation of this mechanism was elucidated. Renal TRPV5 mRNA of NCKX3 KO mice increased in both male and female mice. Expression of TRPV6 mRNA was only down-regulated in the duodenum of male KO mice. Renal- and duodenal expression of PTHR and VDR were not changed; however, GR mRNA expression was increased in the kidney of NCKX3 KO mice. Depletion of the NCKX3 gene in a KO mouse model showed loss of bone mineral contents and increased plasma parathyroid hormone, suggesting that NCKX3 may play a role in regulating calcium homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy.

PubMed

Gu, Junlian; Cheng, Yanli; Wu, Hao; Kong, Lili; Wang, Shudong; Xu, Zheng; Zhang, Zhiguo; Tan, Yi; Keller, Bradley B; Zhou, Honglan; Wang, Yuehui; Xu, Zhonggao; Cai, Lu

2017-02-01

We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM. © 2017 by the American Diabetes Association.

Sexually Dimorphic Role of G Protein-Coupled Estrogen Receptor (GPER) in Modulating Energy Homeostasis

PubMed Central

Davis, Kathryn E.; Carstens, Elizabeth J.; Irani, Boman G.; Gent, Lana M.; Hahner, Lisa M.; Clegg, Deborah J.

2014-01-01

The classical estrogen receptors, estrogen receptor-α and estrogen receptor-β are well established in the regulation of body weight and energy homeostasis in both male and female mice, whereas, the role for G protein-coupled estrogen receptor 1 (GPER) as a modulator of energy homeostasis remains controversial. This study sought to determine whether gene deletion of GPER (GPER KO) alters body weight, body adiposity, food intake, and energy homeostasis in both males and females. Male mice lacking GPER developed moderate obesity and larger adipocyte size beginning at 8 weeks of age, with significant reductions in energy expenditure, but not food intake or adipocyte number. Female GPER KO mice developed increased body weight relative to WT females a full 6 weeks later than the male GPER KO mice. Female GPER KO mice also had reductions in energy expenditure, but not significant increases in body fat content. Consistent with their decrease in energy expenditure, GPER KO males and females showed significant reductions in two brown fat thermogenic proteins. GPER KO females, prior to their divergence in body weight, were less sensitive than WT females to the feeding-inhibitory effects of leptin and CCK. Additionally, body weight was not as modulated by ovariectomy or estradiol replacement in GPER KO mice. Estradiol treatment activated phosphorylated extracellular signal-regulated kinase (pERK) in WT but not GPER KO females. For the first time, GPER expression was found in the adipocyte but not the stromal fraction of adipose tissue. Together, these results provide new information elucidating a sexual dimorphism in GPER function in the development of postpubertal energy balance. PMID:24560890

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

PubMed

Prossnitz, Eric R; Hathaway, Helen J

2015-09-01

Estrogens, predominantly 17β-estradiol, exert diverse effects throughout the body in both normal and pathophysiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERβ) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERβ KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also demonstrated roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer. Copyright © 2015. Published by Elsevier Ltd.

β-Catenin is Essential for Ethanol Metabolism and Protection Against Alcohol-mediated Liver Steatosis in Mice

PubMed Central

Liu, Shiguang; Yeh, Tzu-Hsuan; Singh, Vijay P.; Shiva, Sruti; Krauland, Lindsay; Li, Huanan; Zhang, Pili; Kharbanda, Kusum; Ritov, Vladimir; Monga, Satdarshan P. S.; Scott, Donald K.; Eagon, Patricia K.; Behari, Jaideep

2011-01-01

The liver plays a central role in ethanol metabolism and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pair-wise fashion. Liver histology, biochemistry, and gene expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and five to six-fold higher serum ALT and AST levels. KO mice had modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD-2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetyl cysteine (NAC) did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to co-precipitate with FoxO3, the upstream regulator of SOD-2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were upregulated in ethanol-fed WT mice but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. Conclusion β-catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo. PMID:22031168

Modification of activity-dependent increases in cerebellar blood flow by extracellular potassium in anaesthetized rats

PubMed Central

Caesar, Kirsten; Akgören, Nuran; Mathiesen, Claus; Lauritzen, Martin

1999-01-01

The hypothesis that potassium ions mediate activity-dependent increases of cerebral blood flow was examined in rat cerebellar cortex using ion-selective microelectrodes and laser-Doppler flowmetry. Increases of cerebellar blood flow (CeBF) and extracellular potassium concentration ([K+]o) were evoked by stimulation of parallel fibres and climbing fibres, and by microinjection of KCl into the cortex. For parallel fibre stimulation, there was a maximal increase in [K+]o to 6.3 ± 0.5 mm and in CeBF of 122 ± 11%. Climbing fibre stimulation gave a maximal increase in [K+]o to 4.4 ± 0.2 mm and in CeBF of 157 ± 20%. This indicates different maxima for [K+]o and CeBF, dependent on the afferent system activated. [K+]o and CeBF responses evoked by parallel or climbing fibre stimulation increased rapidly at the onset of stimulation, but exhibited different time courses during the remainder of the stimulation period and during return to baseline. Microinjections of KCl into the cortex increased [K+]o to levels comparable to those evoked by parallel fibre stimulation. The corresponding CeBF increases were the same as, or smaller than, for parallel fibre stimulation, and much smaller than for climbing fibre stimulation. This suggests that mediators other than [K+]o are important for activity-dependent cerebral blood flow increases. The present study showed that increased [K+]o is involved in CeBF regulation in the parallel fibre system, but is of limited importance for CeBF regulation in the climbing fibre system. The hypothesis that K+ is a major mediator of activity-dependent blood flow increases is probably not generally applicable to all brain regions and all types of neuronal stimulation. PMID:10517819

Sexually dimorphic role of G protein-coupled estrogen receptor (GPER) in modulating energy homeostasis.

PubMed

Davis, Kathryn E; Carstens, Elizabeth J; Irani, Boman G; Gent, Lana M; Hahner, Lisa M; Clegg, Deborah J

2014-06-01

This article is part of a Special Issue "Energy Balance". The classical estrogen receptors, estrogen receptor-α and estrogen receptor-β are well established in the regulation of body weight and energy homeostasis in both male and female mice, whereas, the role for G protein-coupled estrogen receptor 1 (GPER) as a modulator of energy homeostasis remains controversial. This study sought to determine whether gene deletion of GPER (GPER KO) alters body weight, body adiposity, food intake, and energy homeostasis in both males and females. Male mice lacking GPER developed moderate obesity and larger adipocyte size beginning at 8 weeks of age, with significant reductions in energy expenditure, but not food intake or adipocyte number. Female GPER KO mice developed increased body weight relative to WT females a full 6 weeks later than the male GPER KO mice. Female GPER KO mice also had reductions in energy expenditure, but no significant increases in body fat content. Consistent with their decrease in energy expenditure, GPER KO males and females showed significant reductions in two brown fat thermogenic proteins. GPER KO females, prior to their divergence in body weight, were less sensitive than WT females to the feeding-inhibitory effects of leptin and CCK. Additionally, body weight was not as modulated by ovariectomy or estradiol replacement in GPER KO mice. Estradiol treatment activated phosphorylated extracellular signal-regulated kinase (pERK) in WT but not GPER KO females. For the first time, GPER expression was found in the adipocyte but not the stromal fraction of adipose tissue. Together, these results provide new information elucidating a sexual dimorphism in GPER function in the development of postpubertal energy balance. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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.

Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

PubMed

Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-Ichi; Igarashi, Kazuhiko; Harata, Masahiko

2017-01-01

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

Elastase‐2, an angiotensin II‐generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction

PubMed Central

Silva, Marcondes A B; Durand, Marina T; Prado, Cibele M; Oliveira, Eduardo B; Ribeiro, Mauricio S; Salgado, Helio C; Salgado, Maria Cristina O; Tostes, Rita C

2017-01-01

Background and Purpose Angiotensin II (Ang II), whose generation largely depends on angiotensin‐converting enzyme (ACE) activity, mediates most of the renin‐angiotensin‐system (RAS) effects. Elastase‐2 (ELA‐2), a chymotrypsin‐serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II‐generation in resistance arteries are unknown. We hypothesized that ELA‐2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Experimental Approach Concentration‐effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA‐2 knockout (ELA‐2KO) mice subjected to left anterior descending coronary artery ligation (MI). Key Results MI size was similar in WT and ELA‐2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA‐2KO mice. Ang I‐induced contractions increased in WT mice subjected to MI (MI‐WT) compared with sham‐WT mice. No differences were observed in Ang I reactivity between arteries from ELA‐2KO and ELA‐2KO subjected to MI (MI‐ELA‐2KO). Ang I contractions increased in arteries from MI‐WT versus MI‐ELA‐2KO mice. Chymostatin attenuated Ang I‐induced vascular contractions in WT mice, but did not affect Ang I responses in ELA‐2KO arteries. Conclusions and Implications These results provide the first evidence that ELA‐2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA‐2 as a key player in ACE‐independent dysregulation of the RAS. PMID:28222221

Elastase-2, an angiotensin II-generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction.

PubMed

Becari, Christiane; Silva, Marcondes A B; Durand, Marina T; Prado, Cibele M; Oliveira, Eduardo B; Ribeiro, Mauricio S; Salgado, Helio C; Salgado, Maria Cristina O; Tostes, Rita C

2017-05-01

Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI). MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries. These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS. © 2017 The British Pharmacological Society.

Somatostatin Is Essential for the Sexual Dimorphism of GH Secretion, Corticosteroid-Binding Globulin Production, and Corticosterone Levels in Mice

PubMed Central

Adams, Jessica M.; Otero-Corchon, Veronica; Hammond, Geoffrey L.; Veldhuis, Johannes D.; Qi, Nathan

2015-01-01

Distinct male and female patterns of pituitary GH secretion produce sexually differentiated hepatic gene expression profiles, thereby influencing steroid and xenobiotic metabolism. We used a fully automated system to obtain serial nocturnal blood samples every 15 minutes from cannulated wild-type (WT) and somatostatin knockout (Sst-KO) mice to determine the role of SST, the principal inhibitor of GH release, in the generation of sexually dimorphic GH pulsatility. WT males had lower mean and median GH values, less random GH secretory bursts, and longer trough periods between GH pulses than WT females. Each of these parameters was feminized in male Sst-KO mice, whereas female Sst-KO mice had higher GH levels than all other groups, but GH pulsatility was unaffected. We next performed hepatic mRNA profiling with high-density microarrays. Male Sst-KO mice exhibited a globally feminized pattern of GH-dependent mRNA levels, but female Sst-KO mice were largely unaffected. Among the differentially expressed female-predominant genes was Serpina6, which encodes corticosteroid-binding globulin (CBG). Increased CBG was associated with elevated diurnal peak plasma corticosterone in unstressed WT females and both sexes of Sst-KO mice compared with WT males. Sst-KO mice also had exaggerated ACTH and corticosterone responses to acute restraint stress. However, consistent with their lack of phenotypic signs of excess glucocorticoids, cerebrospinal fluid concentrations of free corticosterone in Sst-KO mice were not elevated. In summary, SST is necessary for the prolonged interpulse troughs that define masculinized pituitary GH secretion. SST also contributes to sexual dimorphism of the hypothalamic-pituitary-adrenal axis via GH-dependent regulation of hepatic CBG production. PMID:25551181

Conditional disruption of the prolyl hydroxylase domain-containing protein 2 (Phd2) gene defines its key role in skeletal development.

PubMed

Cheng, Shaohong; Xing, Weirong; Pourteymoor, Sheila; Mohan, Subburaman

2014-10-01

We have previously shown that the increase in osterix (Osx) expression during osteoblast maturation is dependent on the activity of the prolyl hydroxylase domain-containing protein 2 (Phd2), a key regulator of protein levels of the hypoxia-inducible factor family proteins in many tissues. In this study, we generated conditional Phd2 knockout mice (cKO) in osteoblast lineage cells by crossing floxed Phd2 mice with a Col1α2-iCre line to investigate the function of Phd2 in vivo. The cKO mice developed short stature and premature death at 12 to 14 weeks of age. Bone mineral content, bone area, and bone mineral density were decreased in femurs and tibias, but not vertebrae of the cKO mice compared to WT mice. The total volume (TV), bone volume (BV), and bone volume fraction (BV/TV) in the femoral trabecular bones of cKO mice were significantly decreased. Cross-sectional area of the femoral mid-diaphysis was also reduced in the cKO mice. The reduced bone size and trabecular bone volume in the cKO mice were a result of impaired bone formation but not bone resorption as revealed by dynamic histomorphometric analyses. Bone marrow stromal cells derived from cKO mice formed fewer and smaller nodules when cultured with mineralization medium. Quantitative RT-PCR and immunohistochemistry detected reduced expression of Osx, osteocalcin, and bone sialoprotein in cKO bone cells. These data indicate that Phd2 plays an important role in regulating bone formation in part by modulating expression of Osx and bone formation marker genes. © 2014 American Society for Bone and Mineral Research.

Direct renin inhibition modulates insulin resistance in caveolin-1-deficient mice

PubMed Central

Chuengsamarn, Somlak; Garza, Amanda E.; Krug, Alexander W.; Romero, Jose R.; Adler, Gail K.; Williams, Gordon H.; Pojoga, Luminita H.

2012-01-01

Objective To test the hypothesis that aliskiren improves the metabolic phenotype in a genetic mouse model of the metabolic syndrome (the caveolin-1 knock out (KO) mouse). Materials/Methods Eleven-week-old cav-1 KO and genetically matched wild-type (WT) mice were randomized to three treatment groups: placebo (n = 8/group), amlodipine (6 mg/kg/day, n = 18/ group), and aliskiren (50 mg/kg/day, n = 18/ group). After three weeks of treatment, all treatment groups were assessed for several measures of insulin resistance (fasting insulin and glucose, HOMA-IR, and the response to an intraperitoneal glucose tolerance test (ipGTT)) as well as for triglyceride levels and the blood pressure response to treatment. Results Treatment with aliskiren did not affect the ipGTT response but significantly lowered the HOMA-IR and insulin levels in cav-1 KO mice. However, treatment with amlodipine significantly degraded the ipGTT response, as well as the HOMA-IR and insulin levels in the cav-1 KO mice. Aliskiren also significantly lowered triglyceride levels in the cav-1 KO but not in the WT mice. Moreover, aliskiren treatment had a significantly greater effect on blood pressure readings in the cav-1 KO vs. WT mice, and marginally more effective than amlodipine. Conclusions Our results support the hypothesis that aliskiren reduces insulin resistance as indicated by improved HOMA-IR in cav-1 KO mice whereas amlodipine treatment resulted in changes consistent with increased insulin resistance. In addition, aliskiren was substantially more effective in lowering blood pressure in the cav-1 KO mouse model than in WT mice and marginally more effective than amlodipine. PMID:22954672

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

PubMed

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.

Endocannabinoid receptor deficiency affects maternal care and alters the dam's hippocampal oxytocin receptor and brain-derived neurotrophic factor expression.

PubMed

Schechter, M; Weller, A; Pittel, Z; Gross, M; Zimmer, A; Pinhasov, A

2013-10-01

Maternal care is the newborn's first experience of social interaction, and this influences infant survival, development and social competences throughout life. We recently found that postpartum blocking of the endocannabinoid receptor-1 (CB1R) altered maternal behaviour. In the present study, maternal care was assessed by the time taken to retrieve pups, pups' ultrasonic vocalisations (USVs) and pup body weight, comparing CB1R deleted (CB1R KO) versus wild-type (WT) mice. After culling on postpartum day 8, hippocampal expression of oxytocin receptor (OXTR), brain-derived neurotrophic factor (BDNF) and stress-mediating factors were evaluated in CB1R KO and WT dams. Comparisons were also performed with nulliparous (NP) CB1R KO and WT mice. Compared to WT, CB1R KO dams were slower to retrieve their pups. Although the body weight of the KO pups did not differ from the weight of WT pups, they emitted fewer USVs. This impairment of the dam-pup relationship correlated with a significant reduction of OXTR mRNA and protein levels among CB1R KO dams compared to WT dams. Furthermore, WT dams exhibited elevated OXTR mRNA expression, as well as increased levels of mineralocorticoid and glucocorticoid receptors, compared to WT NP mice. By contrast, CB1R KO dams showed no such elevation of OXTR expression, alongside lower BDNF and mineralocorticoid receptors, as well as elevated corticotrophin-releasing hormone mRNA levels, when compared to CB1R KO NP. Thus, it appears that the disruption of endocannabinoid signalling by CB1R deletion alters expression of the OXTR, apparently leading to deleterious effects upon maternal behaviour. © 2013 British Society for Neuroendocrinology.

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

PubMed

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.

Methylprednisolone prevents nerve injury-induced hyperalgesia in neprilysin knockout mice.

PubMed

He, Lan; Uçeyler, Nurcan; Krämer, Heidrun H; Colaço, Maria Nandini; Lu, Bao; Birklein, Frank; Sommer, Claudia

2014-03-01

The pathophysiology of the complex regional pain syndrome involves enhanced neurogenic inflammation mediated by neuropeptides. Neutral endopeptidase (neprilysin, NEP) is a key enzyme in neuropeptide catabolism. Our previous work revealed that NEP knock out (ko) mice develop more severe hypersensitivity to thermal and mechanical stimuli after chronic constriction injury (CCI) of the sciatic nerve than wild-type (wt) mice. Because treatment with glucocorticoids is effective in early complex regional pain syndrome, we investigated whether methylprednisolone (MP) reduces pain and sciatic nerve neuropeptide content in NEP ko and wt mice with nerve injury. After CCI, NEP ko mice developed more severe thermal and mechanical hypersensitivity and hind paw edema than wt mice, confirming previous findings. Hypersensitivity was prevented by MP treatment in NEP ko but not in wt mice. MP treatment had no effect on protein levels of calcitonin-gene related peptide, substance P, and bradykinin in sciatic nerves of NEP ko mice. Endothelin-1 (ET-1) levels were higher in naïve and nerve-injured NEP ko than in wt mice, without an effect of MP treatment. Gene expression of the ET-1 receptors ETAR and ETBR was not different between genotypes and was not altered after CCI, but was increased after additional MP treatment. The ETBR agonist IRL-1620 was analgesic in NEP ko mice after CCI, and the ETBR antagonist BQ-788 showed a trend to reduce the analgesic effect of MP. The results provide evidence that MP reduces CCI-induced hyperalgesia in NEP ko mice, and that this may be related to ET-1 via analgesic actions of ETBR. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Immature morphological properties in subcellular-scale structures in the dentate gyrus of Schnurri-2 knockout mice: a model for schizophrenia and intellectual disability.

PubMed

Nakao, Akito; Miyazaki, Naoyuki; Ohira, Koji; Hagihara, Hideo; Takagi, Tsuyoshi; Usuda, Nobuteru; Ishii, Shunsuke; Murata, Kazuyoshi; Miyakawa, Tsuyoshi

2017-12-12

Accumulating evidence suggests that subcellular-scale structures such as dendritic spine and mitochondria may be involved in the pathogenesis/pathophysiology of schizophrenia and intellectual disability. Previously, we proposed mice lacking Schnurri-2 (Shn2; also called major histocompatibility complex [MHC]-binding protein 2 [MBP-2], or human immunodeficiency virus type I enhancer binding protein 2 [HIVEP2]) as a schizophrenia and intellectual disability model with mild chronic inflammation. In the mutants' brains, there are increases in C4b and C1q genes, which are considered to mediate synapse elimination during postnatal development. However, morphological properties of subcellular-scale structures such as dendritic spine in Shn2 knockout (KO) mice remain unknown. In this study, we conducted three-dimensional morphological analyses in subcellular-scale structures in dentate gyrus granule cells of Shn2 KO mice by serial block-face scanning electron microscopy. Shn2 KO mice showed immature dendritic spine morphology characterized by increases in spine length and decreases in spine diameter. There was a non-significant tendency toward decrease in spine density of Shn2 KO mice over wild-type mice, and spine volume was indistinguishable between genotypes. Shn2 KO mice exhibited a significant reduction in GluR1 expression and a nominally significant decrease in SV2 expression, while PSD95 expression had a non-significant tendency to decrease in Shn2 KO mice. There were significant decreases in dendrite diameter, nuclear volume, and the number of constricted mitochondria in the mutants. Additionally, neuronal density was elevated in Shn2 KO mice. These results suggest that Shn2 KO mice serve as a unique tool for investigating morphological abnormalities of subcellular-scale structures in schizophrenia, intellectual disability, and its related disorders.

The High Calcium, High Phosphorus Rescue Diet Is Not Suitable to Prevent Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

PubMed

Grundmann, Sarah M; Brandsch, Corinna; Rottstädt, Daniela; Kühne, Hagen; Stangl, Gabriele I

2017-01-01

The vitamin D receptor (VDR) knockout (KO) mouse is a common model to unravel novel metabolic functions of vitamin D. It is recommended to feed these mice a high calcium (2%), high phosphorus (1.25%) diet, termed rescue diet (RD) to prevent hypocalcaemia and secondary hyperparathyroidism. First, we characterized the individual response of VDR KO mice to feeding a RD and found that the RD was not capable of normalizing the parathyroid hormone (PTH) concentrations in each VDR KO mouse. In a second study, we aimed to study whether RD with additional 1 and 2% calcium (in total 3 and 4% of the diet) is able to prevent secondary hyperparathyroidism in the VDR KO mice. Wild type (WT) mice and VDR KO mice that received a normal calcium and phosphorus diet (ND) served as controls. Data demonstrated that the RD was no more efficient than the ND in normalizing PTH levels. An excessive dietary calcium concentration of 4% was required to reduce serum PTH concentrations in the VDR KO mice to PTH levels measured in WT mice. This diet, however, resulted in higher concentrations of circulating intact fibroblast growth factor 23 (iFGF23). To conclude, the commonly used RD is not suitable to normalize the serum PTH in VDR KO mice. Extremely high dietary calcium concentrations are necessary to prevent secondary hyperthyroidism in these mice, with the consequence that iFGF23 concentrations are being raised. Considering that PTH and iFGF23 exert numerous VDR independent effects, data obtained from VDR KO mice cannot be attributed solely to vitamin D.

Loss of Sirt3 Limits Bone Marrow Cell-Mediated Angiogenesis and Cardiac Repair in Post-Myocardial Infarction

PubMed Central

Zeng, Heng; Li, Lanfang; Chen, Jian-Xiong

2014-01-01

Sirtuin-3 (Sirt3) has a critical role in the regulation of human aging and reactive oxygen species (ROS) formation. A recent study has identified Sirt3 as an essential regulator of stem cell aging. This study investigated whether Sirt3 is necessary for bone marrow cell (BMC)-mediated cardiac repair in post-myocardial infarction (MI). In vitro, BMC-derived endothelial progenitor cells (EPCs) from wild type (WT) and Sirt3KO mice were cultured. EPC angiogenesis, ROS formation and apoptosis were assessed. In vivo, WT and Sirt3 KO mice were subjected to MI and BMCs from WT and Sirt3 KO mice were injected into ischemic area immediately. The expression of VEGF and VEGFR2 was reduced in Sirt3KO-EPCs. Angiogenic capacities and colony formation were significantly impaired in Sirt3KO-EPCs compared to WT-EPCs. Loss of Sirt3 further enhanced ROS formation and apoptosis in EPCs. Overexpression of Sirt3 or treatment with NADPH oxidase inhibitor apocynin (Apo, 200 and 400 microM) rescued these abnormalities. In post-MI mice, BMC treatment increased number of Sca1+/c-kit+ cells; enhanced VEGF expression and angiogenesis whereas Sirt3KO-BMC treatment had little effects. BMC treatment also attenuated NADPH oxidase subunits p47phox and gp91phox expression, and significantly reduced ROS formation, apoptosis, fibrosis and hypertrophy in post-MI mice. Sirt3KO-BMC treatment did not display these beneficial effects. In contrast, Sirt3KO mice treated with BMCs from WT mice attenuated myocardial apoptosis, fibrosis and improved cardiac function. Our data demonstrate that Sirt3 is essential for BMC therapy; and loss of Sirt3 limits BMC-mediated angiogenesis and cardiac repair in post-MI. PMID:25192254

Effects of deletion of ER-alpha in osteoblast-lineage cells on bone mass and adaptation to mechanical loading differs in female and male mice

PubMed Central

Melville, Katherine M.; Kelly, Natalie H.; Surita, Gina; Buchalter, Daniel B.; Schimenti, John C.; Main, Russell P.; Ross, F. Patrick; van der Meulen, Marjolein C. H.

2015-01-01

Estrogen receptor alpha (ERα) has been implicated in bone’s response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell-specific ERαKO mice. Therefore, we crossed ERα floxed (ERαfl/fl) and osteocalcin-Cre (OC-Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO) and littermate controls (LC). At 10 weeks of age the left tibia was loaded in vivo for two weeks. We analyzed bone mass through microCT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared to LC, female pOC-ERαKO mice had decreased cortical and cancellous bone mass, while male pOC-ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC-ERαKO female L5 vertebrae, and with increased maximum moment in pOC-ERαKO male femora. Female pOC-ERαKO mice responded more to mechanical loading, while the response of pOC-ERαKO male animals was similar to their littermate controls. PMID:25707500

Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1

PubMed Central

Tanner, Colby B.; Madsen, Steven R.; Hallowell, David M.; Goring, Darren M. J.; Moore, Timothy M.; Hardman, Shalene E.; Heninger, Megan R.; Atwood, Daniel R.

2013-01-01

LKB1 and its downstream targets of the AMP-activated protein kinase family are important regulators of many aspects of skeletal muscle cell function, including control of mitochondrial content and capillarity. LKB1 deficiency in skeletal and cardiac muscle (mLKB1-KO) greatly impairs exercise capacity. However, cardiac dysfunction in that genetic model prevents a clear assessment of the role of skeletal muscle LKB1 in the observed effects. Our purposes here were to determine whether skeletal muscle-specific knockout of LKB1 (skmLKB1-KO) decreases exercise capacity and mitochondrial protein content, impairs accretion of mitochondrial proteins after exercise training, and attenuates improvement in running performance after exercise training. We found that treadmill and voluntary wheel running capacity was reduced in skmLKB1-KO vs. control (CON) mice. Citrate synthase activity, succinate dehydrogenase activity, and pyruvate dehydrogenase kinase content were lower in KO vs. CON muscles. Three weeks of treadmill training resulted in significantly increased treadmill running performance in both CON and skmLKB1-KO mice. Citrate synthase activity increased significantly with training in both genotypes, but protein content and activity for components of the mitochondrial electron transport chain increased only in CON mice. Capillarity and VEGF protein was lower in skmLKB1-KO vs. CON muscles, but VEGF increased with training only in skmLKB1-KO. Three hours after an acute bout of muscle contractions, PGC-1α, cytochrome c, and VEGF gene expression all increased in CON but not skmLKB1-KO muscles. Our findings indicate that skeletal muscle LKB1 is required for accretion of some mitochondrial proteins but not for early exercise capacity improvements with exercise training. PMID:23982155

Natriuretic Peptide Receptor Guanylyl Cyclase-A in Podocytes is Renoprotective but Dispensable for Physiologic Renal Function

PubMed Central

Staffel, Janina; Valletta, Daniela; Federlein, Anna; Ehm, Katharina; Volkmann, Regine; Füchsl, Andrea M.; Witzgall, Ralph; Kuhn, Michaela

2017-01-01

The cardiac natriuretic peptides (NPs), atrial NP and B-type NP, regulate fluid homeostasis and arterial BP through renal actions involving increased GFR and vascular and tubular effects. Guanylyl cyclase-A (GC-A), the transmembrane cGMP-producing receptor shared by these peptides, is expressed in different renal cell types, including podocytes, where its function is unclear. To study the effects of NPs on podocytes, we generated mice with a podocyte-specific knockout of GC-A (Podo-GC-A KO). Despite the marked reduction of GC-A mRNA in GC-A KO podocytes to 1% of the control level, Podo-GC-A KO mice and control littermates did not differ in BP, GFR, or natriuresis under baseline conditions. Moreover, infusion of synthetic NPs similarly increased the GFR and renal perfusion in both genotypes. Administration of the mineralocorticoid deoxycorticosterone-acetate (DOCA) in combination with high salt intake induced arterial hypertension of similar magnitude in Podo-GC-A KO mice and controls. However, only Podo-GC-A KO mice developed massive albuminuria (controls: 35-fold; KO: 5400-fold versus baseline), hypoalbuminemia, reduced GFR, and marked glomerular damage. Furthermore, DOCA treatment led to decreased expression of the slit diaphragm-associated proteins podocin, nephrin, and synaptopodin and to enhanced transient receptor potential canonical 6 (TRPC6) channel expression and ATP-induced calcium influx in podocytes of Podo-GC-A KO mice. Concomitant treatment of Podo-GC-A KO mice with the TRPC channel blocker SKF96365 markedly ameliorated albuminuria and glomerular damage in response to DOCA. In conclusion, the physiologic effects of NPs on GFR and natriuresis do not involve podocytes. However, NP/GC-A/cGMP signaling protects podocyte integrity under pathologic conditions, most likely by suppression of TRPC channels. PMID:27153922

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

PubMed

Toyoda, Hiromi; Honda, Yoshiko; Tanaka, Susumu; Miyagawa, Taku; Honda, Makoto; Honda, Kazuki; Tokunaga, Katsushi; Kodama, Tohru

2017-01-01

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.

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

PubMed

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.

Hybridization capture reveals evolution and conservation across the entire Koala retrovirus genome.

PubMed

Tsangaras, Kyriakos; Siracusa, Matthew C; Nikolaidis, Nikolas; Ishida, Yasuko; Cui, Pin; Vielgrader, Hanna; Helgen, Kristofer M; Roca, Alfred L; Greenwood, Alex D

2014-01-01

The koala retrovirus (KoRV) is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus) to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin.

Hyperactivity and lack of social discrimination in the adolescent Fmr1 knockout mouse.

PubMed

Sørensen, Emilie M; Bertelsen, Freja; Weikop, Pia; Skovborg, Maria M; Banke, Tue; Drasbek, Kim R; Scheel-Krüger, Jørgen

2015-12-01

The aims of this study were to investigate behaviour relevant to human autism spectrum disorder (ASD) and the fragile X syndrome in adolescent Fmr1 knockout (KO) mice and to evaluate the tissue levels of striatal monoamines. Fmr1 KO mice were evaluated in the open field, marble burying and three-chamber test for the presence of hyperactivity, anxiety, repetitive behaviour, sociability and observation of social novelty compared with wild-type (WT) mice. The Fmr1 KO mice expressed anxiety and hyperactivity in the open field compared with WT mice. This increased level of hyperactivity was confirmed in the three-chamber test. Fmr1 KO mice spent more time with stranger mice compared with the WT. However, after a correction for hyperactivity, their apparent increase in sociability became identical to that of the WT. Furthermore, the Fmr1 KO mice could not differentiate between a familiar or a novel mouse. Monoamines were measured by HPLC: Fmr1 KO mice showed an increase in the striatal dopamine level. We conclude that the fragile X syndrome model seems to be useful for understanding certain aspects of ASD and may have translational interest for studies of social behaviour when hyperactivity coexists in ASD patients.

Hybridization Capture Reveals Evolution and Conservation across the Entire Koala Retrovirus Genome

PubMed Central

Ishida, Yasuko; Cui, Pin; Vielgrader, Hanna; Helgen, Kristofer M.; Roca, Alfred L.; Greenwood, Alex D.

2014-01-01

The koala retrovirus (KoRV) is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus) to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin. PMID:24752422

Telmisartan regresses left ventricular hypertrophy in caveolin-1 deficient mice

PubMed Central

Kreiger, Marta H; Di Lorenzo, Annarita; Teutsch, Christine; Kauser, Katalin; Sessa, William C.

2011-01-01

The role of angiotensin II (Ang II) in promoting cardiac hypertrophy is well known, however the role of the Ang II in a spontaneous model of hypertrophy in mice lacking the protein caveolin-1 (Cav- KO) has not been explored. In this study, WT and Cav-1 KO mice were treated with angiotensin receptor blocker (ARB), telmisartan, and cardiac function assessed by echocardiography. Treatment of Cav-1 KO mice with telmisartan significantly improved cardiac function compared to age-matched, vehicle treated Cav-1 KO mice, while telmisartan did not affected cardiac function in WT mice. Both left ventricular (LV) weight to body weight ratios and LV to tibial length ratios were also reverted by telmisartan in Cav-1 KO but not WT mice. LV hypertrophy was associated with increased expression of natriuretic peptides-A and –B, β-myosin heavy chain and TGF-β and telmisartan treatment normalized the expression of these genes. Telmisartan reduced the expression of collagen genes (Col1A and Col3A) and associated perivascular fibrosis in intramyocardial vessels in Cav-1 KO mice. In conclusion, telmisartan treatment reduces indexes of cardiac hypertrophy in this unique genetic model of spontaneous LV hypertrophy. PMID:20585312

Gal knockout and beyond.

PubMed

Zhong, R

2007-01-01

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

Functionally improved bone in Calbindin-D28k knockout mice

PubMed Central

Margolis, David S.; Kim, Devin; Szivek, John A.; Lai, Li-Wen; Lien, Yeong-Hau H.

2008-01-01

In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, μCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% ± 3.1) compared to wild-type (WT) mice (45.4% ± 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm ± 0.278 in Calbindin-D28k KO mice, and 4.046 mm ± 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. μCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% ± 3.1) compared to WT mice (24.7% ± 4.9) which was primarily due to increased trabecular number (8.99 mm−1 ± 0.94 in Calbindin-D28k KO mice compared to 6.75 mm−1 ± 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 μstrain/kg ± 1266) compared to WT mice (6973 μstrain/kg ± 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N ± 2.1) compared to WT mice (15.0 N ± 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling. PMID:16631426

Role of corticosteroid binding globulin in emotional reactivity sex differences in mice.

PubMed

Minni, A M; de Medeiros, G F; Helbling, J C; Duittoz, A; Marissal-Arvy, N; Foury, A; De Smedt-Peyrusse, V; Pallet, V; Moisan, M P

2014-12-01

Sex differences exist for stress reactivity as well as for the prevalence of depression, which is more frequent in women of reproductive age and often precipitated by stressful events. In animals, the differential effect of stress on male's and female's emotional behavior has been well documented. Crosstalk between the gonadal and stress hormones, in particular between estrogens and glucocorticoids, underlie these sex differences on stress vulnerability. We have previously shown that corticosteroid binding globulin (CBG) deficiency in a mouse model (Cbg k.o.) leads, in males, to an increased despair-like behavior caused by suboptimal corticosterone stress response. Because CBG displays a sexual dimorphism and is regulated by estrogens, we have now investigated whether it plays a role in the sex differences observed for emotional reactivity in mice. By analyzing Cbg k.o. and wild-type (WT) animals of both sexes, we detected sex differences in despair-like behavior in WT mice but not in Cbg k.o. animals. We showed through ovariectomy and estradiol (E2) replacement that E2 levels explain the sex differences found in WT animals. However, the manipulation of E2 levels did not affect the emotional behavior of Cbg k.o. females. As Cbg k.o. males, Cbg k.o. females have markedly reduced corticosterone levels across the circadian cycle and also after stress. Plasma free corticosterone levels in Cbg k.o. mice measured immediately after stress were blunted in both sexes compared to WT mice. A trend for higher mean levels of ACTH in Cbg k.o. mice was found for both sexes. The turnover of a corticosterone bolus was increased in Cbg k.o. Finally, the glucocorticoid-regulated immediate early gene early growth response 1 (Egr1) showed a blunted mRNA expression in the hippocampus of Cbg k.o. mutants while mineralocorticoid and glucocorticoid receptors presented sex differences but equivalent mRNA expression between genotypes. Thus, in our experimental conditions, sex differences for despair-like behavior in WT mice are explained by estrogens levels. Also, in both sexes, the presence of CBG is required to attain optimal glucocorticoid concentrations and normal emotional reactivity, although in females this is apparent only under low E2 concentrations. These findings suggest a complex interaction of CBG and E2 on emotional reactivity in females. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fish oil and krill oil supplementations differentially regulate lipid catabolic and synthetic pathways in mice

PubMed Central

2014-01-01

Background Marine derived oils are rich in long-chain polyunsaturated omega-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have long been associated with health promoting effects such as reduced plasma lipid levels and anti-inflammatory effects. Krill oil (KO) is a novel marine oil on the market and is also rich in EPA and DHA, but the fatty acids are incorporated mainly into phospholipids (PLs) rather than triacylglycerols (TAG). This study compares the effects of fish oil (FO) and KO on gene regulation that influences plasma and liver lipids in a high fat diet mouse model. Methods Male C57BL/6J mice were fed either a high-fat diet (HF) containing 24% (wt/wt) fat (21.3% lard and 2.3% soy oil), or the HF diet supplemented with FO (15.7% lard, 2.3% soy oil and 5.8% FO) or KO (15.6% lard, 2.3% soy oil and 5.7% KO) for 6 weeks. Total levels of cholesterol, TAG, PLs, and fatty acid composition were measured in plasma and liver. Gene regulation was investigated using quantitative PCR in liver and intestinal epithelium. Results Plasma cholesterol (esterified and unesterified), TAG and PLs were significantly decreased with FO. Analysis of the plasma lipoprotein particles indicated that the lipid lowering effect by FO is at least in part due to decreased very low density lipoprotein (VLDL) content in plasma with subsequent liver lipid accumulation. KO lowered plasma non-esterified fatty acids (NEFA) with a minor effect on fatty acid accumulation in the liver. In spite of a lower omega-3 fatty acid content in the KO supplemented diet, plasma and liver PLs omega-3 levels were similar in the two groups, indicating a higher bioavailability of omega-3 fatty acids from KO. KO more efficiently decreased arachidonic acid and its elongation/desaturation products in plasma and liver. FO mainly increased the expression of several genes involved in fatty acid metabolism, while KO specifically decreased the expression of genes involved in the early steps of isoprenoid/cholesterol and lipid synthesis. Conclusions The data show that both FO and KO promote lowering of plasma lipids and regulate lipid homeostasis, but with different efficiency and partially via different mechanisms. PMID:24834104

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

PubMed

Denton, P W; Nochi, T; Lim, A; Krisko, J F; Martinez-Torres, F; Choudhary, S K; Wahl, A; Olesen, R; Zou, W; Di Santo, J P; Margolis, D M; Garcia, J V

2012-09-01

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

PubMed Central

Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

2013-01-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function.

PubMed

Back, Steven H; Adapala, Naga Suresh; Barbe, Mary F; Carpino, Nick C; Tsygankov, Alexander Y; Sanjay, Archana

2013-04-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers

PubMed Central

Pozzi, Benedetta; Amodio, Stefania; Lucano, Caterina; Sciullo, Anna; Ronzoni, Simona; Castelletti, Daniela; Adler, Thure; Treise, Irina; Betsholtz, Ingrid Holmberg; Rathkolb, Birgit; Busch, Dirk H.; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Betsholtz, Christer; Casola, Stefano; Di Fiore, Pier Paolo; Offenhäuser, Nina

2012-01-01

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220+ bone marrow cells, CD19− thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis. PMID:23226392

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

PubMed Central

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

2010-01-01

In rodent species, sexual differentiation of the brain for many reproductive processes depends largely on estradiol. This was recently confirmed again by using the α-fetoprotein knockout (AFP-KO) mouse model, which lacks the protective actions of α-fetoprotein against maternal estradiol and as a result represents a good model to determine the contribution of prenatal estradiol to the sexual differentiation of the brain and behavior. Female AFP-KO mice were defeminized and masculinized with regard to their neuroendocrine responses as well as sexual behavior. Since parental behavior is also strongly sexually differentiated in mice, we used the AFP-KO mouse model here to ask whether parental responses are differentiated prenatally under the influence of estradiol. It was found that AFP-KO females showed longer latencies to retrieve pups to the nest and also exhibited lower levels of crouching over the pups in the nest in comparison to WT females. In fact, they resembled males (WT and AFP-KO). Other measures of maternal behavior, for example the incidence of infanticide, tended to be higher in AFP-KO females than in WT females but this increase failed to reach statistical significance. The deficits observed in parental behavior of AFP-KO females could not be explained by any changes in olfactory function, novelty recognition or anxiety. Thus our results suggest that prenatal estradiol defeminizes the parental brain in mice. PMID:20109458

Elevated body temperature during sleep in orexin knockout mice

PubMed Central

Mochizuki, Takatoshi; Klerman, Elizabeth B.; Sakurai, Takeshi; Scammell, Thomas E.

2008-01-01

Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is thought to promote heat production during wakefulness, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, the Tb of orexin KO mice was 0.4°C higher than wild-type (WT) littermates during the dark period. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During sustained wakefulness, Tb was the same in both groups. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0°C, but Tb in orexin KO mice decreased only 0.4°C. Even during intense recovery sleep after 8 hr of sleep deprivation, the Tb of orexin KO mice remained 0.7°C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy. PMID:16556901

Alterations in bladder function associated with urothelial defects in uroplakin II and IIIa knockout mice.

PubMed

Aboushwareb, Tamer; Zhou, Ge; Deng, Fang-Ming; Turner, Chanda; Andersson, Karl-Erik; Tar, Moses; Zhao, Weixin; Melman, Arnold; D'Agostino, Ralph; Sun, Tung-Tien; Christ, George J

2009-01-01

The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/dysfunction were studied in male and female wild type and knockout (KO) mice. UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types. These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO's, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former. These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction.

Development of a model for predicting reaction rate constants of organic chemicals with ozone at different temperatures.

PubMed

Li, Xuehua; Zhao, Wenxing; Li, Jing; Jiang, Jingqiu; Chen, Jianji; Chen, Jingwen

2013-08-01

To assess the persistence and fate of volatile organic compounds in the troposphere, the rate constants for the reaction with ozone (kO3) are needed. As kO3 values are only available for hundreds of compounds, and experimental determination of kO3 is costly and time-consuming, it is of importance to develop predictive models on kO3. In this study, a total of 379 logkO3 values at different temperatures were used to develop and validate a model for the prediction of kO3, based on quantum chemical descriptors, Dragon descriptors and structural fragments. Molecular descriptors were screened by stepwise multiple linear regression, and the model was constructed by partial least-squares regression. The cross validation coefficient QCUM(2) of the model is 0.836, and the external validation coefficient Qext(2) is 0.811, indicating that the model has high robustness and good predictive performance. The most significant descriptor explaining logkO3 is the BELm2 descriptor with connectivity information weighted atomic masses. kO3 increases with increasing BELm2, and decreases with increasing ionization potential. The applicability domain of the proposed model was visualized by the Williams plot. The developed model can be used to predict kO3 at different temperatures for a wide range of organic chemicals, including alkenes, cycloalkenes, haloalkenes, alkynes, oxygen-containing compounds, nitrogen-containing compounds (except primary amines) and aromatic compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inflammatory pain-induced signaling events following a conditional deletion of the N-methyl-D-aspartate receptor in spinal cord dorsal horn.

PubMed

Cheng, H T; Suzuki, M; Hegarty, D M; Xu, Q; Weyerbacher, A R; South, S M; Ohata, M; Inturrisi, C E

2008-08-26

The N-methyl-d-aspartate (NMDA) receptor in the spinal cord dorsal horn (SCDH) is one of the mechanisms involved in central sensitization during chronic pain. Previously, this laboratory created a spatio-temporal knockout (KO) of the N-methyl-d-aspartate receptor I (NR1) subunit in the mouse SCDH. The NR1 KO completely blocks NR1 gene and subsequent NMDA receptor expression and function in SCDH neurons. In the NR1 KO mice, the mechanical and cold allodynia induced at 24 h after complete Freund's adjuvant (CFA) was reduced. However, the protective effects of KO were transient and were not seen at 48 h after CFA. These observations suggest the presence of NMDA-independent pathways that contribute to CFA-induced pain. CFA induces the activation of several signaling cascades in the SCDH, including protein kinase C (PKC)gamma and extracellular signal-regulated kinases (ERK1/2). The phosphorylation of PKCgamma and ERK1/2 was inhibited in the SCDH of NR1 KO mice up to 48 h after CFA treatment, suggesting that these pathways are NMDA receptor-dependent. Interestingly, neuronal cyclooxygenase (COX) -2 expression and microglial p38 phosphorylation were induced in the SCDH of the NR1 KO at 48 h after CFA. Our findings provide evidence that inflammatory reactions are responsible for the recurrence of pain after NR1 KO in the SCDH.

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

PubMed

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.

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

PubMed Central

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

The Role of Epithelial Sodium Channel ENaC and the Apical Cl-/HCO3- Exchanger Pendrin in Compensatory Salt Reabsorption in the Setting of Na-Cl Cotransporter (NCC) Inactivation.

PubMed

Patel-Chamberlin, Mina; Varasteh Kia, Mujan; Xu, Jie; Barone, Sharon; Zahedi, Kamyar; Soleimani, Manoocher

2016-01-01

The absence of NCC does not cause significant salt wasting in NCC deficient mice under basal conditions. We hypothesized that ENaC and pendrin play important roles in compensatory salt absorption in the setting of NCC inactivation, and their inhibition and/or downregulation can cause significant salt wasting in NCC KO mice. WT and NCC KO mice were treated with a daily injection of either amiloride, an inhibitor of ENaC, or acetazolamide (ACTZ), a blocker of salt and bicarbonate reabsorption in the proximal tubule and an inhibitor of carbonic anhydrases in proximal tubule and intercalated cells, or a combination of acetazolamide plus amiloride for defined durations. Animals were subjected to daily balance studies. At the end of treatment, kidneys were harvested and examined. Blood samples were collected for electrolytes and acid base analysis. Amiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p<0.03, n = 4) but caused only a slight change in UO in WT mice (p>0.05). The increase in UO in NCC KO mice was associated with a significant increase in sodium excretion (from 0.25 mmol/24 hrs at baseline to 0.35 mmol/24 hrs after amiloride injection, p<0.05, n = 4). Daily treatment with ACTZ for 6 days resulted in >80% reduction of kidney pendrin expression in both WT and NCC KO mice. However, ACTZ treatment noticeably increased urine output and salt excretion only in NCC KO mice (with urine output increasing from a baseline of 1.1 ml/day to 2.3 ml/day and sodium excretion increasing from 0.22 mmole/day before to 0.31 mmole/day after ACTZ) in NCC KO mice; both parameters were significantly higher than in WT mice. Western blot analysis demonstrated significant enhancement in ENaC expression in medulla and cortex of NCC KO and WT mice in response to ACTZ injection for 6 days, and treatment with amiloride in ACTZ-pretreated mice caused a robust increase in salt excretion in both NCC KO and WT mice. Pendrin KO mice did not display a significant increase in urine output or salt excretion after treatment with amiloride or ACTZ. 1. ENaC plays an important role in salt reabsorption in NCC KO mice. 2. NCC contributes to compensatory salt reabsorption in the setting of carbonic anhydrase inhibition, which is associated with increased delivery of salt from the proximal tubule and the down regulation of pendrin. 3. ENaC is upregulated by ACTZ treatment and its inhibition by amiloride causes significant diuresis in NCC KO and WT mice. Despite being considered mild agents individually, we propose that the combination of acetazolamide and amiloride in the setting of NCC inhibition (i.e., hydrochlorothiazide) will be a powerful diuretic regimen.

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

PubMed Central

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

2011-01-01

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 over-representation 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 over-representation 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. PMID:21803530

Anti-Trichomonas vaginalis properties of the oil of Amomum tsao-ko and its major component, geraniol.

PubMed

Dai, Min; Peng, Cheng; Peng, Fu; Xie, Chengbin; Wang, Pinjia; Sun, Fenghui

2016-01-01

Trichomonosis, caused by the flagellate protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease (STD) and 5-nitroimidazole drugs are used for the treatment. However, a growing number of T. vaginalis isolates are resistant to these drugs, which make it becomes an urgent issue. The current study was designed to evaluate the anti-T. vaginalis activity of the essential oil from A. tsao-ko used in traditional Chinese medicine and as a spice and its main component, geraniol. The anti-T. vaginalis activities of A. tsao-ko essential oil and geraniol were evaluated by the minimum lethal concentration (MLC) and 50% inhibitory concentration (IC50) in vitro. The morphological changes of T. vaginalis were observed by transmission electron microscopy (TEM). Additionally, sub-MLC concentration treatment with sub-MLC A. tsao-ko essential oil and geraniol was also performed. This study shows that MLC/IC50 of A. tsao-ko essential oil was 44.97 µg/ml/22.49 µg/ml for T. vaginalis isolate Tv1, and 89.93 µg/ml/44.97 µg/ml for T. vaginalis isolate Tv2. Those of geraniol were 342.96 µg/ml/171.48 µg/ml, respectively. After A. tsao-ko essential oil or geraniol treatment, obvious similar morphological changes of T. vaginalis were observed by TEM: the nuclear membrane was damaged, nuclei were dissolved, and the chromatin was accumulated; in the cytoplasm, numerous vacuoles appeared, rough endoplasmic reticulum dilated, the number of ribosomes were reduced, organelles disintegrated, the cell membrane was partially damaged, with cytoplasmic leakage, and cell disintegration was observed. The action time did not increase the effect of A. tsao-ko essential oil or geraniol against T. vaginalis, as no significant difference was observed after sub-MLC concentration treatment for 1, 3, and 5 h with A. tsao-ko essential oil and geraniol. The study describes the first report on the activity and morphological changes of A. tsao-ko essential oil and geraniol against T. vaginalis. The results obtained herein presented new opportunities for antitrichomonal drugs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Min Sook; Woo, Min-Yeong; Department of Biomedical Sciences, The Graduate School, Ajou University

2014-10-01

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

Epileptogenesis following Kainic Acid-Induced Status Epilepticus in Cyclin D2 Knock-Out Mice with Diminished Adult Neurogenesis

PubMed Central

Kondratiuk, Ilona; Plucinska, Gabriela; Miszczuk, Diana; Wozniak, Grazyna; Szydlowska, Kinga; Kaczmarek, Leszek; Filipkowski, Robert K.; Lukasiuk, Katarzyna

2015-01-01

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2 – 10 days) in wt mice and 8 days (range 2 – 16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1 – 3.4; cD2 KO: 0.57, range 0.1 – 2.0 seizures/day) or median seizure duration (wt: 51 s, range 23 – 103; cD2 KO: 51 s, range 23 – 103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state. PMID:26020770

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

PubMed

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.

Detection of kobuvirus RNA in Japanese domestic dogs.

PubMed

Soma, Takehisa; Matsubayashi, Makoto; Sasai, Kazumi

2016-12-01

To investigate whether kokuvirus is present in Japanese dogs, we examined the fecal samples obtained from 94 diarrheal household dogs and 50 clinically healthy kenneled dogs by RT-PCR. The gene was detected in 37.2% and 48.0% in the former and the latter, respectively, suggesting that canine kobuvirus (CaKoV) is circulating among Japanese dogs. From the result of the latter, however, CaKoV may not be a primary pathogen. Furthermore, all gene-positive dogs were purebreds aged four months or younger. This finding suggests that CaKoV endemic is confined in multi-dog environments, and the dogs have a strong age-dependent resistance to CaKoV.

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

PubMed Central

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

Cluster and principal component analysis based on SSR markers of Amomum tsao-ko in Jinping County of Yunnan Province

NASA Astrophysics Data System (ADS)

Ma, Mengli; Lei, En; Meng, Hengling; Wang, Tiantao; Xie, Linyan; Shen, Dong; Xianwang, Zhou; Lu, Bingyue

2017-08-01

Amomum tsao-ko is a commercial plant that used for various purposes in medicinal and food industries. For the present investigation, 44 germplasm samples were collected from Jinping County of Yunnan Province. Clusters analysis and 2-dimensional principal component analysis (PCA) was used to represent the genetic relations among Amomum tsao-ko by using simple sequence repeat (SSR) markers. Clustering analysis clearly distinguished the samples groups. Two major clusters were formed; first (Cluster I) consisted of 34 individuals, the second (Cluster II) consisted of 10 individuals, Cluster I as the main group contained multiple sub-clusters. PCA also showed 2 groups: PCA Group 1 included 29 individuals, PCA Group 2 included 12 individuals, consistent with the results of cluster analysis. The purpose of the present investigation was to provide information on genetic relationship of Amomum tsao-ko germplasm resources in main producing areas, also provide a theoretical basis for the protection and utilization of Amomum tsao-ko resources.

Genetic deletion of the norepinephrine transporter decreases vulnerability to seizures

PubMed Central

Kaminski, Rafal M.; Shippenberg, Toni S.; Witkin, Jeffrey M.; Rocha, Beatriz A.

2005-01-01

Norepinephrine (NE) has been reported to modulate neuronal excitability and act as endogenous anticonvulsant. In the present study we used NE transporter knock-out mice (NET-KO), which are characterized by high levels of extracellular NE, to investigate the role of endogenous NE in seizure susceptibility. Seizure thresholds for cocaine (i.p.), pentylenetetrazol (i.v.) and kainic acid (i.v.) were compared in NET-KO, heterozygous (NET-HT) and wild type (NET-WT) female mice. The dose-response curve for cocaine-induced convulsions was significantly shifted to the right in NET-KO mice, indicating higher seizure thresholds. The threshold doses of pentylenetetrazol that induced clonic and tonic seizures were also significantly higher in NET-KO when compared to NET-WT mice. Similarly, NET-KO mice displayed higher resistance to convulsions engendered by kainic acid. For all drugs tested, the response of NET-HT mice was always intermediate. These data provide further support for a role of endogenous NE in the control of seizure susceptibility. PMID:15911120

Desnutrin/ATGL Activates PPARδ to Promote Mitochondrial Function for Insulin Secretion in Islet β cells

PubMed Central

Tang, Tianyi; Abbott, Marcia J.; Ahmadian, Maryam; Lopes, Andressa B.; Wang, Yuhui; Sul, Hei Sook

2013-01-01

Excessive caloric intake leading to obesity is associated with insulin resistance and dysfuntion of islet β cells. High fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (βKO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. βKO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPARδ target genes involved in mitochondrial oxidation. Furthermore, synthetic PPARδ, but not PPARα, agonist restores GSIS and expression of mitochondrial oxidative genes in βKO mice, revealing desnutrin-catalyzed lipolysis generates PPARδ ligands. Finally, adenoviral expression of desnutrin in βKO islets restores all defects of βKO islet phenotype and function including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPARδ-mitochondrial oxidation axis in regulating islet β cell GSIS. PMID:24268737

Ko Displacement Theory for Structural Shape Predictions

NASA Technical Reports Server (NTRS)

Ko, William L.

2010-01-01

The development of the Ko displacement theory for predictions of structure deformed shapes was motivated in 2003 by the Helios flying wing, which had a 247-ft (75-m) wing span with wingtip deflections reaching 40 ft (12 m). The Helios flying wing failed in midair in June 2003, creating the need to develop new technology to predict in-flight deformed shapes of unmanned aircraft wings for visual display before the ground-based pilots. Any types of strain sensors installed on a structure can only sense the surface strains, but are incapable to sense the overall deformed shapes of structures. After the invention of the Ko displacement theory, predictions of structure deformed shapes could be achieved by feeding the measured surface strains into the Ko displacement transfer functions for the calculations of out-of-plane deflections and cross sectional rotations at multiple locations for mapping out overall deformed shapes of the structures. The new Ko displacement theory combined with a strain-sensing system thus created a revolutionary new structure- shape-sensing technology.

Canine kobuvirus infections in Korean dogs.

PubMed

Oem, Jae-Ku; Choi, Jeong-Won; Lee, Myoung-Heon; Lee, Kyoung-Ki; Choi, Kyoung-Seong

2014-10-01

To investigate canine kobuvirus (CaKoV) infection, fecal samples (n = 59) were collected from dogs with or without diarrhea (n = 21 and 38, respectively) in the Republic of Korea (ROK) in 2012. CaKoV infection was detected in four diarrheic samples (19.0 %) and five non-diarrheic samples (13.2 %). All CaKoV-positive dogs with diarrhea were found to be infected in mixed infections with canine distemper virus and canine parvovirus or canine adenovirus. There was no significant difference in the prevalence of CaKoV in dogs with and without diarrhea. By phylogenetic analysis based on partial 3D genes and complete genome sequences, the Korean isolates were found to be closely related to each other regardless of whether they were associated with diarrhea, and to the canine kobuviruses identified in the USA and UK. This study supports the conclusion that CaKoVs from different countries are not restricted geographically and belong to a single lineage.

Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

PubMed Central

Wang, Huaishan; Yang, Jia; Yang, Qi; Fu, Yi; Hu, Yu; Liu, Fang; Wang, Weiqing; Cui, Lianxian; Chen, Hui; Zhang, Jianmin; He, Wei

2016-01-01

Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis. PMID:27907096

Mucin deficiency causes functional and structural changes of the ocular surface.

PubMed

Floyd, Anne M; Zhou, Xu; Evans, Christopher; Rompala, Olivia J; Zhu, Lingxiang; Wang, Mingwu; Chen, Yin

2012-01-01

MUC5AC is the most abundant gel-forming mucin in the ocular system. However, the specific function is unknown. In the present study, a Muc5ac knockout (KO) mouse model was subject to various physiological measurements as compared to its wide-type (WT) control. Interestingly, when KO mice were compared to WT mice, the mean tear break up time (TBUT) values were significantly lower and corneal fluorescein staining scores were significantly higher. But the tear volume was not changed. Despite the lack of Muc5ac expression in the conjunctiva of KO mice, Muc5b expression was significantly increased in these mice. Corneal opacification, varying in location and severity, was found in a few KO mice but not in WT mice. The present results suggest a significant difference in the quality, but not the quantity, of tear fluid in the KO mice compared to WT mice. Dry eye disease is multifactorial and therefore further evaluation of the varying components of the tear film, lacrimal unit and corneal structure of these KO mice may help elucidate the role of mucins in dry eye disease. Because Muc5ac knockout mice have clinical features of dry eye, this mouse model will be extremely useful for further studies regarding the pathophysiology of the ocular surface in dry eye in humans.

CD4 T cells promote rather than control tuberculosis in the absence of PD-1-mediated inhibition.

PubMed

Barber, Daniel L; Mayer-Barber, Katrin D; Feng, Carl G; Sharpe, Arlene H; Sher, Alan

2011-02-01

Although CD4 T cells are required for host resistance to Mycobacterium tuberculosis, they may also contribute to pathology. In this study, we examine the role of the inhibitory receptor PD-1 and its ligand PD-L1 during M. tuberculosis infection. After aerosol exposure, PD-1 knockout (KO) mice develop high numbers of M. tuberculosis-specific CD4 T cells but display markedly increased susceptibility to infection. Importantly, we show that CD4 T cells themselves drive the increased bacterial loads and pathology seen in infected PD-1 KO mice, and PD-1 deficiency in CD4 T cells is sufficient to trigger early mortality. PD-L1 KO mice also display enhanced albeit less severe susceptibility, indicating that T cells are regulated by multiple PD ligands during M. tuberculosis infection. M. tuberculosis-specific CD8 T cell responses were normal in PD-1 KO mice, and CD8 T cells only had a minor contribution to the exacerbated disease in the M. tuberculosis-infected PD-1 KO and PD-L1 KO mice. Thus, in the absence of the PD-1 pathway, M. tuberculosis benefits from CD4 T cell responses, and host resistance requires inhibition by PD-1 to prevent T cell-driven exacerbation of the infection.

Ca2+-Binding Protein 1 Regulates Hippocampal-dependent Memory and Synaptic Plasticity.

PubMed

Yang, Tian; Britt, Jeremiah K; Cintrón-Pérez, Coral J; Vázquez-Rosa, Edwin; Tobin, Kevin V; Stalker, Grant; Hardie, Jason; Taugher, Rebecca J; Wemmie, John; Pieper, Andrew A; Lee, Amy

2018-06-01

Ca 2+ -binding protein 1 (CaBP1) is a Ca 2+ -sensing protein similar to calmodulin that potently regulates voltage-gated Ca 2+ channels. Unlike calmodulin, however, CaBP1 is mainly expressed in neuronal cell-types and enriched in the hippocampus, where its function is unknown. Here, we investigated the role of CaBP1 in hippocampal-dependent behaviors using mice lacking expression of CaBP1 (C-KO). By western blot, the largest CaBP1 splice variant, caldendrin, was detected in hippocampal lysates from wild-type (WT) but not C-KO mice. Compared to WT mice, C-KO mice exhibited mild deficits in spatial learning and memory in both the Barnes maze and in Morris water maze reversal learning. In contextual but not cued fear-conditioning assays, C-KO mice showed greater freezing responses than WT mice. In addition, the number of adult-born neurons in the hippocampus of C-KO mice was ∼40% of that in WT mice, as measured by bromodeoxyuridine labeling. Moreover, hippocampal long-term potentiation was significantly reduced in C-KO mice. We conclude that CaBP1 is required for cellular mechanisms underlying optimal encoding of hippocampal-dependent spatial and fear-related memories. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age*

PubMed Central

Gorski, Jeff P.; Huffman, Nichole T.; Vallejo, Julian; Brotto, Leticia; Chittur, Sridar V.; Breggia, Anne; Stern, Amber; Huang, Jian; Mo, Chenglin; Seidah, Nabil G.; Bonewald, Lynda; Brotto, Marco

2016-01-01

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10–12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss. PMID:26719336

Glial degeneration with oxidative damage drives neuronal demise in MPSII disease

PubMed Central

Zalfa, Cristina; Verpelli, Chiara; D'Avanzo, Francesca; Tomanin, Rosella; Vicidomini, Cinzia; Cajola, Laura; Manara, Renzo; Sala, Carlo; Scarpa, Maurizio; Vescovi, Angelo Luigi; De Filippis, Lidia

2016-01-01

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the iduronate 2-sulfatase (IDS) enzyme, causing progressive neurodegeneration in patients. Neural stem cells (NSCs) derived from the IDS-ko mouse can recapitulate MPSII pathogenesis in vitro. In differentiating IDS-ko NSCs and in the aging IDS-ko mouse brain, glial degeneration precedes neuronal degeneration. Here we show that pure IDS-ko NSC-derived astrocytes are selectively able to drive neuronal degeneration when cocultured with healthy neurons. This phenotype suggests concurrent oxidative damage with metabolic dysfunction. Similar patterns were observed in murine IDS-ko animals and in human MPSII brains. Most importantly, the mutant phenotype of IDS-ko astrocytes was reversed by low oxygen conditions and treatment with vitamin E, which also reversed the toxic effect on cocultured neurons. Moreover, at very early stages of disease we detected in vivo the development of a neuroinflammatory background that precedes astroglial degeneration, thus suggesting a novel model of MPSII pathogenesis, with neuroinflammation preceding glial degeneration, which is finally followed by neuronal death. This hypothesis is also consistent with the progression of white matter abnormalities in MPSII patients. Our study represents a novel breakthrough in the elucidation of MPSII brain pathogenesis and suggests the antioxidant molecules as potential therapeutic tools to delay MPSII onset and progression. PMID:27512952

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

PubMed Central

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

2016-01-01

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

Combined behavioral studies and in vivo imaging of inflammatory response and expression of mGlu5 receptors in schnurri-2 knockout mice.

PubMed

Choi, Ji-Kyung; Zhu, Aijun; Jenkins, Bruce G; Hattori, Satoko; Kil, Kun-Eek; Takagi, Tsuyoshi; Ishii, Shunsuke; Miyakawa, Tsuyoshi; Brownell, Anna-Liisa

2015-11-16

Schnurri-2 (Shn-2) knockout (KO) mice have been proposed as a preclinical neuroinflammatory schizophrenia model. We used behavioral studies and imaging markers that can be readily translated to human populations to explore brain effects of inflammation. Shn-2 KO mice and their littermate control mice were imaged with two novel PET ligands; an inflammation marker [(11)C]PBR28 and the mGluR5 ligand [(18)F]FPEB. Locomotor activity was measured using open field exploration with saline, methamphetamine or amphetamine challenge. A significantly increased accumulation of [(11)C]PBR28 was found in the cortex, striatum, hippocampus and olfactory bulb of Shn-2 KO mice. Increased mGluR5 binding was also observed in the cortex and hippocampus of the Shn-2 KO mice. Open field locomotor testing revealed a large increase in novelty-induced hyperlocomotion in Shn-2 KO mice with abnormal (decreased) responses to either methamphetamine or amphetamine. These data provide additional support to demonstrate that the Shn-2 KO mouse model exhibits several behavioral and pathological markers resembling human schizophrenia making it an attractive translational model for the disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A krill oil supplemented diet suppresses hepatic steatosis in high-fat fed rats.

PubMed

Ferramosca, Alessandra; Conte, Annalea; Burri, Lena; Berge, Kjetil; De Nuccio, Francesco; Giudetti, Anna Maria; Zara, Vincenzo

2012-01-01

Krill oil (KO) is a dietary source of n-3 polyunsaturated fatty acids, mainly represented by eicosapentaenoic acid and docosahexaenoic acid bound to phospholipids. The supplementation of a high-fat diet with 2.5% KO efficiently prevented triglyceride and cholesterol accumulation in liver of treated rats. This effect was accompanied by a parallel reduction of the plasma levels of triglycerides and glucose and by the prevention of a plasma insulin increase. The investigation of the molecular mechanisms of KO action in high-fat fed animals revealed a strong decrease in the activities of the mitochondrial citrate carrier and of the cytosolic acetyl-CoA carboxylase and fatty acid synthetase, which are both involved in hepatic de novo lipogenesis. In these animals a significant increase in the activity of carnitine palmitoyl-transferase I and in the levels of carnitine was also observed, suggesting a concomitant stimulation of hepatic fatty acid oxidation. The KO supplemented animals also retained an efficient mitochondrial oxidative phosphorylation, most probably as a consequence of a KO-induced arrest of the uncoupling effects of a high-fat diet. Lastly, the KO supplementation prevented an increase in body weight, as well as oxidative damage of lipids and proteins, which is often found in high-fat fed animals.

A Krill Oil Supplemented Diet Suppresses Hepatic Steatosis in High-Fat Fed Rats

PubMed Central

Ferramosca, Alessandra; Conte, Annalea; Burri, Lena; Berge, Kjetil; De Nuccio, Francesco; Giudetti, Anna Maria; Zara, Vincenzo

2012-01-01

Krill oil (KO) is a dietary source of n-3 polyunsaturated fatty acids, mainly represented by eicosapentaenoic acid and docosahexaenoic acid bound to phospholipids. The supplementation of a high-fat diet with 2.5% KO efficiently prevented triglyceride and cholesterol accumulation in liver of treated rats. This effect was accompanied by a parallel reduction of the plasma levels of triglycerides and glucose and by the prevention of a plasma insulin increase. The investigation of the molecular mechanisms of KO action in high-fat fed animals revealed a strong decrease in the activities of the mitochondrial citrate carrier and of the cytosolic acetyl-CoA carboxylase and fatty acid synthetase, which are both involved in hepatic de novo lipogenesis. In these animals a significant increase in the activity of carnitine palmitoyl-transferase I and in the levels of carnitine was also observed, suggesting a concomitant stimulation of hepatic fatty acid oxidation. The KO supplemented animals also retained an efficient mitochondrial oxidative phosphorylation, most probably as a consequence of a KO-induced arrest of the uncoupling effects of a high-fat diet. Lastly, the KO supplementation prevented an increase in body weight, as well as oxidative damage of lipids and proteins, which is often found in high-fat fed animals. PMID:22685607

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy.

PubMed

Melegari, Irene; Sarchese, Vittorio; Di Profio, Federica; Robetto, Serena; Carella, Emanuele; Bermudez Sanchez, Sandra; Orusa, Riccardo; Martella, Vito; Marsilio, Fulvio; Di Martino, Barbara

2018-02-01

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D'Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.

Nepro is localized in the nucleolus and essential for preimplantation development in mice.

PubMed

Hashimoto, Masakazu; Sato, Tatsuya; Muroyama, Yuko; Fujimura, Lisa; Hatano, Masahiko; Saito, Tetsuichiro

2015-09-01

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2-cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria-associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus-associated protein, and its loss leads to the apoptosis before blastocyst formation in mice. © 2015 Japanese Society of Developmental Biologists.

Hyperactivity and depression-like traits in Bax KO mice

PubMed Central

Krahe, Thomas E.; Medina, Alexandre E.; Lantz, Crystal L.; Filgueiras, Cláudio C.

2018-01-01

The Bax gene is a member of the Bcl-2 gene family and its pro-apoptotic Bcl-associated X (Bax) protein is believed to be crucial in regulating apoptosis during neuronal development as well as following injury. With the advent of mouse genomics, mice lacking the pro-apoptotic Bax gene (Bax KO) have been extensively used to study how cell death helps to determine synaptic circuitry formation during neurodevelopment and disease. Surprisingly, in spite of its wide use and the association of programmed neuronal death with motor dysfunctions and depression, the effects of Bax deletion on mice spontaneous locomotor activity and depression-like traits are unknown. Here we examine the behavioral characteristics of Bax KO male mice using classical paradigms to evaluate spontaneous locomotor activity and depressive-like responses. In the open field, Bax KO animals exhibited greater locomotor activity than their control littermates. In the forced swimming test, Bax KO mice displayed greater immobility times, a behavior despair state, when compared to controls. Collectively, our findings corroborate the notion that a fine balance between cell survival and death early during development is critical for normal brain function later in life. Furthermore, it points out the importance of considering depressive-like and hyperactivity behavioral phenotypes when conducting neurodevelopmental and other studies using the Bax KO strain. PMID:26363094

Glial degeneration with oxidative damage drives neuronal demise in MPSII disease.

PubMed

Zalfa, Cristina; Verpelli, Chiara; D'Avanzo, Francesca; Tomanin, Rosella; Vicidomini, Cinzia; Cajola, Laura; Manara, Renzo; Sala, Carlo; Scarpa, Maurizio; Vescovi, Angelo Luigi; De Filippis, Lidia

2016-08-11

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the iduronate 2-sulfatase (IDS) enzyme, causing progressive neurodegeneration in patients. Neural stem cells (NSCs) derived from the IDS-ko mouse can recapitulate MPSII pathogenesis in vitro. In differentiating IDS-ko NSCs and in the aging IDS-ko mouse brain, glial degeneration precedes neuronal degeneration. Here we show that pure IDS-ko NSC-derived astrocytes are selectively able to drive neuronal degeneration when cocultured with healthy neurons. This phenotype suggests concurrent oxidative damage with metabolic dysfunction. Similar patterns were observed in murine IDS-ko animals and in human MPSII brains. Most importantly, the mutant phenotype of IDS-ko astrocytes was reversed by low oxygen conditions and treatment with vitamin E, which also reversed the toxic effect on cocultured neurons. Moreover, at very early stages of disease we detected in vivo the development of a neuroinflammatory background that precedes astroglial degeneration, thus suggesting a novel model of MPSII pathogenesis, with neuroinflammation preceding glial degeneration, which is finally followed by neuronal death. This hypothesis is also consistent with the progression of white matter abnormalities in MPSII patients. Our study represents a novel breakthrough in the elucidation of MPSII brain pathogenesis and suggests the antioxidant molecules as potential therapeutic tools to delay MPSII onset and progression.

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

PubMed

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.

Induction of alternative proinflammatory cytokines accounts for sustained psoriasiform skin inflammation in IL-17C+IL-6KO mice

PubMed Central

Fritz, Yi; Klenotic, Philip A.; Swindell, William R.; Yin, ZhiQiang; Groft, Sarah G.; Zhang, Li; Baliwag, Jaymie; Camhi, Maya I.; Diaconu, Doina; Young, Andrew B.; Foster, Alexander M.; Johnston, Andrew; Gudjonsson, Johann E.; McCormick, Thomas S.; Ward, Nicole L.

2016-01-01

IL-6 inhibition has been unsuccessful in treating psoriasis, despite high levels of tissue and serum IL-6 in patients. Additionally, de novo psoriasis onset has been reported following IL-6 blockade in rheumatoid arthritis patients. To explore mechanisms underlying these clinical observations, we backcrossed an established psoriasiform mouse model (IL-17C+ mice) with IL-6 deficient mice (IL-17C+KO) and examined the cutaneous phenotype. IL-17C+KO mice initially exhibited decreased skin inflammation, however this decrease was transient and reversed rapidly, concomitant with increases in skin Tnf, Il36α/β/γ, Il24, Epgn and S100a8/a9 to levels higher than those found in IL-17C+ mice. Comparison of IL-17C+ and IL-17C+KO mouse skin transcriptomes with that of human psoriasis skin, revealed significant correlation among transcripts of psoriasis patient skin and IL-17C+KO mouse skin, and confirmed an exacerbation of the inflammatory signature in IL-17C+KO mice that aligns closely with human psoriasis. Transcriptional analyses of IL-17C+ and IL-17C+KO primary keratinocytes confirmed increased expression of proinflammatory molecules, suggesting that in the absence of IL-6, keratinocytes increase production of numerous additional proinflammatory cytokines. These preclinical findings may provide insight into why arthritis patients being treated with IL-6 inhibitors develop new onset psoriasis and why IL-6 blockade for the treatment of psoriasis has not been clinically effective. PMID:27984037

Contributions of Interleukin‐33 and TSLP in a papain‐soaked contact lens‐induced mouse conjunctival inflammation model

PubMed Central

Sugita, Jobu; Asada, Yosuke; Ishida, Waka; Iwamoto, Satoshi; Sudo, Katsuko; Suto, Hajime; Matsunaga, Toru; Fukuda, Ken; Fukushima, Atsuki; Yokoi, Norihiko; Ohno, Tatsukuni; Azuma, Miyuki; Ebihara, Nobuyuki; Saito, Hirohisa; Kubo, Masato; Nakae, Susumu

2017-01-01

Abstract Introduction Pathological changes of severe chronic allergic conjunctivitis are driven not only via acquired immunity but also via innate immunity. Type 2 immune response‐initiating cytokines may play some roles as innate immunity‐dependent components of the ocular surface inflammation. To investigate the involvement of type 2 immune response‐initiating cytokines in innate immunity‐dependent, papain‐induced conjunctival inflammation model using IL‐25‐, IL‐33‐, and TSLP receptor (TSLPR)‐knockout (KO) mice with reference to basophils and ILC2. Methods Papain‐soaked contact lenses (papain‐CLs) were installed in the conjunctival sacs of C57BL/6‐IL‐25 KO, IL‐33 KO, TSLPR KO, Rag2 KO, Bas‐TRECK, and wild‐type mice and their eyes were sampled at day 5. The eosinophil and basophil infiltration in papain‐CL model was evaluated histologically and cytokine expression was examined. To clarify the roles of basophils and ILC2, basophil/ILC2‐depletion experiments were carried out. Results Papain‐induced conjunctival inflammation exhibited eosinophil infiltration and upregulation of Th2 cytokine expression. Reduction of eosinophil and basophil infiltration and attenuated Th2 cytokine expression were observed in the papain‐CL model using IL‐33 KO and TSLPR KO mice. Depletion of basophils or ILC2s in the conjunctivae of the papain‐CL model reduced eosinophil infiltration. Conclusions Innate immunity‐driven type 2 immune responses of the ocular surface are dependent on IL‐33, TSLP, basophils, and ILC2. These components may be possible therapeutic targets for refractory allergic keratoconjunctivitis. PMID:28730605

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haque, Rizwanul; Umstead, Todd M.; Ponnuru, Padmavathi

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, andmore » 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.« less

Epinephrine increases contextual learning through activation of peripheral β2-adrenoceptors.

PubMed

Alves, Ester; Lukoyanov, Nikolay; Serrão, Paula; Moura, Daniel; Moreira-Rodrigues, Mónica

2016-06-01

Phenylethanolamine-N-methyltransferase knockout (Pnmt-KO) mice are unable to synthesize epinephrine and display reduced contextual fear. However, the precise mechanism responsible for impaired contextual fear learning in these mice is unknown. Our aim was to study the mechanism of epinephrine-dependent contextual learning. Wild-type (WT) or Pnmt-KO (129x1/SvJ) mice were submitted to a fear conditioning test either in the absence or in the presence of epinephrine, isoprenaline (non-selective β-adrenoceptor agonist), fenoterol (selective β2-adrenoceptor agonist), epinephrine plus sotalol (non-selective β-adrenoceptor antagonist), and dobutamine (selective β1-adrenoceptor agonist). Catecholamines were separated by reverse-phase HPLC and quantified by electrochemical detection. Blood glucose was measured by coulometry. Re-exposure to shock context induced higher freezing in WT and Pnmt-KO mice treated with epinephrine and fenoterol than in mice treated with vehicle. In addition, freezing response in Pnmt-KO mice was much lower than in WT mice. Freezing induced by epinephrine was blocked by sotalol in Pnmt-KO mice. Epinephrine and fenoterol treatment restored glycemic response in Pnmt-KO mice. Re-exposure to shock context did not induce a significant difference in freezing in Pnmt-KO mice treated with dobutamine and vehicle. Aversive memories are best retained if moderately high plasma epinephrine concentrations occur at the same moment as the aversive stimulus. In addition, epinephrine increases context fear learning by acting on peripheral β2-adrenoceptors, which may induce high levels of blood glucose. Since glucose crosses the blood-brain barrier, it may enhance hippocampal-dependent contextual learning.

Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein, a key player in Parkinson's disease.

PubMed

Xiao, Wenbin; Shameli, Afshin; Harding, Clifford V; Meyerson, Howard J; Maitta, Robert W

2014-11-01

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response. Copyright © 2014 Elsevier GmbH. All rights reserved.

Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse

PubMed Central

Sourial, Mary; Doering, Laurie C.

2016-01-01

An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. Highlights: • We studied the proliferation of neural stem and progenitor cells in Fragile X. • We examined the role of astrocyte-secreted factors in neural precursor cell biology. • Astrocyte-secreted factors with differential expression in Fragile X identified. PMID:27242437

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokohira, Masanao; Arnold, Lora L.; Pennington, Karen L.

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{sup III}). During the first week of As{sup III} exposure, As3mt KO mice exhibited severe and lethal systemic toxicity. At termination,more » 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{sup 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{sup III} on the bladder epithelium, showing that altered kinetic and dynamic behavior of arsenic can affect its toxicity.« less