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Sample records for prp knock-out mice

  1. Bex1 knock out mice show altered skeletal muscle regeneration

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  2. Motor Deficits and Cerebellar Atrophy in Elovl5 Knock Out Mice.

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    Hoxha, Eriola; Gabriele, Rebecca M C; Balbo, Ilaria; Ravera, Francesco; Masante, Linda; Zambelli, Vanessa; Albergo, Cristian; Mitro, Nico; Caruso, Donatella; Di Gregorio, Eleonora; Brusco, Alfredo; Borroni, Barbara; Tempia, Filippo

    2017-01-01

    Spino-Cerebellar-Ataxia type 38 (SCA38) is caused by missense mutations in the very long chain fatty acid elongase 5 gene, ELOVL5 . The main clinical findings in this disease are ataxia, hyposmia and cerebellar atrophy. Mice in which Elovl5 has been knocked out represent a model of the loss of function hypothesis of SCA38. In agreement with this hypothesis, Elovl5 knock out mice reproduced the main symptoms of patients, motor deficits at the beam balance test and hyposmia. The cerebellar cortex of Elovl5 knock out mice showed a reduction of thickness of the molecular layer, already detectable at 6 months of age, confirmed at 12 and 18 months. The total perimeter length of the Purkinje cell (PC) layer was also reduced in Elovl5 knock out mice. Since Elovl5 transcripts are expressed by PCs, whose dendrites are a major component of the molecular layer, we hypothesized that an alteration of their dendrites might be responsible for the reduced thickness of this layer. Reconstruction of the dendritic tree of biocytin-filled PCs, followed by Sholl analysis, showed that the distribution of distal dendrites was significantly reduced in Elovl5 knock out mice. Dendritic spine density was conserved. These results suggest that Elovl5 knock out mice recapitulate SCA38 symptoms and that their cerebellar atrophy is due, at least in part, to a reduced extension of PC dendritic arborization.

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  4. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

    2005-01-01

    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  5. Reduced hypoxic ventilatory response in newborn mice knocked-out for the progesterone receptor.

    Science.gov (United States)

    Potvin, Catherine; Rossignol, Orlane; Uppari, NagaPraveena; Dallongeville, Arnaud; Bairam, Aida; Joseph, Vincent

    2014-11-01

    Recent studies showed that progesterone stimulates the hypoxic ventilatory response and may reduce apnoea frequency in newborn rats, but so far we still do not know by what mechanisms and whether endogenous progesterone might contribute to respiratory control in neonates. We therefore determined the role of the nuclear progesterone receptor (PR; member of the steroid receptor superfamily) by using wild-type (WT) and PR knock-out (PRKO) mice at postnatal days (P) 1, 4 and 10. We measured the hypoxic ventilatory response (14 and 12% O2, 20 min each) and apnoea frequency in both male and female mice by using whole-body plethysmography. In response to hypoxia, WT male mice had a marked hypoxic ventilatory response at P1 and P10, but not at P4. At P1 and P10, PRKO male mice had a lower hypoxic ventilatory response than WT males. Wild-type female mice had a marked hypoxic ventilatory response at P10, but not at P1 and P4. At P1 and P10, PRKO female mice had a lower hypoxic ventilatory response than WT females. In basal conditions, apnoea frequency was similar in WT and PRKO mice at P1, P4 and P10. During hypoxia, apnoea frequency was higher in WT male mice compared with PRKO male mice and WT female mice at P1. We conclude that PR is a key contributor to the hypoxic ventilatory response in newborn mice, but PR deletion does not increase the frequency of apnoea during normoxia or hypoxia. © 2014 The Authors. Experimental Physiology © 2014 The Physiological Society.

  6. Activation of PPARγ Ameliorates Spatial Cognitive Deficits through Restoring Expression of AMPA Receptors in Seipin Knock-Out Mice.

    Science.gov (United States)

    Zhou, Libin; Chen, Tingting; Li, Guoxi; Wu, Chaoming; Wang, Conghui; Li, Lin; Sha, Sha; Chen, Lei; Liu, George; Chen, Ling

    2016-01-27

    A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Here, we show that seipin deficiency in hippocampal CA1 pyramidal cells caused the reduction of peroxisome proliferator-activated receptor gamma (PPARγ). Twelve-week-old systemic seipin knock-out mice and neuronal seipin knock-out (seipin-nKO) mice, but not adipose seipin knock-out mice, exhibited spatial cognitive deficits as assessed by the Morris water maze and Y-maze, which were ameliorated by the treatment with the PPARγ agonist rosiglitazone (rosi). In addition, seipin-nKO mice showed the synaptic dysfunction and the impairment of NMDA receptor-dependent LTP in hippocampal CA1 regions. The density of AMPA-induced current (IAMPA) in CA1 pyramidal cells and GluR1/GluR2 expression were significantly reduced in seipin-nKO mice, whereas the NMDA-induced current (INMDA) and NR1/NR2 expression were not altered. Rosi treatment in seipin-nKO mice could correct the decrease in expression and activity of AMPA receptor (AMPAR) and was accompanied by recovered synaptic function and LTP induction. Furthermore, hippocampal ERK2 and CREB phosphorylation in seipin-nKO mice were reduced and this could be rescued by rosi treatment. Rosi treatment in seipin-nKO mice elevated BDNF concentration. The MEK inhibitor U0126 blocked rosi-restored AMPAR expression and LTP induction in seipin-nKO mice, but the Trk family inhibitor K252a did not. These findings indicate that the neuronal seipin deficiency selectively suppresses AMPAR expression through reducing ERK-CREB activities, leading to the impairment of LTP and spatial memory, which can be rescued by PPARγ activation. Congenital generalized lipodystrophy 2 (CGL2), caused by loss-of-function mutation of seipin gene, is characterized by mental retardation. By the generation of systemic or neuronal seipin knock-out mice, the present study provides in vivo evidence that neuronal seipin

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  9. Remyelination in experimentally demyelinated connexin 32 KnockOut mice Remielinização em camundongos KnockOut para conexina 32 desmielinizados experimentalmente

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    Adriano Tony Ramos

    2009-06-01

    Full Text Available The aim of this study was to evaluate the role of connexin 32 (Cx 32 during remyelination of the peripheral nervous system, through a local injection of either 0,1% ethidium bromide solution or saline in the sciatic nerve of Cx 32 knockout mice. Euthanasia was performed ranging from 1, 2, 3, 7, 15, 21 to 30 days after injection. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopical techniques were used to analyze the development of the lesions. Within the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compactness and redundant loops (tomacula were conspicuous. We concluded that the regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other laboratory species. Therefore, these results suggest that absence of Cx 32 did not interfere with the normal pattern of remyelination in this model in young mice.Este estudo visou avaliar o papel da conexina 32 (Cx 32 durante a remielinização no sistema nervoso periférico. Uma injeção local de 0,1% de solução de brometo de etídio foi realizada no nervo ciático de camundongos deletados para a Cx 32, com eutanásia dos animais aos 1, 2, 3, 7, 15, 21 e 30 dias pós-injeção. Avaliações histoquímicas, imunoistoquímicas, por imunofluorescência e por microscopia eletrônica de transmissão foram utilizadas na análise do desenvolvimento das lesões. Nos nervos ciáticos, células de Schwann mostraram inicialmente sinais de intoxicação e rejeitaram suas bainhas. Após sete dias, observaram-se finas bainhas neoformadas, com compactação desigual e alças redundantes (tomácula. Conclui-se que a regeneração de bainhas de mielina perdidas no SNP seguiu o padrão já relatado deste modelo em outras espécies de laboratório. Portanto, estes resultados sugerem que a ausência da Cx 32 n

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

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    Malur, Anagha; Huizar, Isham [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Wells, Greg [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Barna, Barbara P. [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Malur, Achut G. [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Thomassen, Mary Jane, E-mail: thomassenm@ecu.edu [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States)

    2011-11-18

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  12. Lethal Zika Virus Disease Models in Young and Older Interferon α/β Receptor Knock Out Mice

    Directory of Open Access Journals (Sweden)

    Andrea Marzi

    2018-04-01

    Full Text Available The common small animal disease models for Zika virus (ZIKV are mice lacking the interferon responses, but infection of interferon receptor α/β knock out (IFNAR−/− mice is not uniformly lethal particularly in older animals. Here we sought to advance this model in regard to lethality for future countermeasure efficacy testing against more recent ZIKV strains from the Asian lineage, preferably the American sublineage. We first infected IFNAR−/− mice subcutaneously with the contemporary ZIKV-Paraiba strain resulting in predominantly neurological disease with ~50% lethality. Infection with ZIKV-Paraiba by different routes established a uniformly lethal model only in young mice (4-week old upon intraperitoneal infection. However, intraperitoneal inoculation of ZIKV-French Polynesia resulted in uniform lethality in older IFNAR−/− mice (10–12-weeks old. In conclusion, we have established uniformly lethal mouse disease models for efficacy testing of antivirals and vaccines against recent ZIKV strains representing the Asian lineage.

  13. Motor and memory testing of long-lived pregnancy-associated plasma protein--a knock-out mice.

    Science.gov (United States)

    Mason, Emily J; Grell, Jacquelyn A; West, Sally A; Conover, Cheryl A

    2014-12-01

    Mice deficient in pregnancy-associated plasma protein-A (PAPP-A), an IGF binding protein protease, have been shown to be resistant to experimentally induced atherosclerosis and diabetic nephropathy, and, in the laboratory environment, live 30-40% longer than wild-type littermates in association with delayed incidence and occurrence of age-related neoplasms and degenerative diseases. PAPP-A is highly expressed in the cerebellum and hippocampus of the mouse brain. Therefore, the studies presented here were aimed at determining motor behavior, learning and retention in PAPP-A knock-out (KO) mice compared to wild-type (WT) littermates with age. Balance and coordination were assessed using an accelerating rotarod; learning and memory were assessed in a Stone T-maze. Time on the rotarod decreased with age but there was no significant difference between PAPP-A KO and WT mice at any of the testing ages. Latency to reach the goal box and number of errors committed in the Stone T-maze did not change with age and there were no significant differences between PAPP-A KO and WT mice. Lack of PAPP-A in mice did not impact central regulation of coordination, learning or memory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Emotional instability but intact spatial cognition in adenosine receptor 1 knock out mice.

    Science.gov (United States)

    Lang, Undine E; Lang, Florian; Richter, Kerstin; Vallon, Volker; Lipp, Hans-Peter; Schnermann, Jürgen; Wolfer, David P

    2003-10-17

    Several lines of evidence point to the involvement of adenosine in the regulation of important central mechanisms such as cognition, arousal, aggression and anxiety. In order to elucidate the involvement of the adenosine A1 receptor (A1AR) in spatial learning and the control of exploratory behaviour, we assessed A1AR knockout mice (A1AR-/-) and their wild-type littermates (A1AR+/+) in a place navigation task in the water maze and in a battery of forced and free exploration tests. In the water maze, A1AR-/- mice showed normal escape latencies and were indistinguishable from controls with respect to measures of spatial performance during both training and probe trial. But despite normal performance they showed increased wall hugging, most prominently after the relocation of the goal platform for reversal training. Quantitative analysis of strategy choices indicated that wall hugging was increased mainly at the expense of chaining and passive floating, whereas the frequency of trials characterised as direct swims or focal searching was normal in A1AR-/- mice. These results indicate intact spatial cognition, but mildly altered emotional reactions to the water maze environment. In line with this interpretation, A1AR-/- mice showed normal levels and patterns of activity, but a mild increase of some measures of anxiety in our battery of forced and free exploration paradigms. These results are in line with findings published using a genetically similar line, but demonstrate that the magnitude of the changes and the range of affected behavioural measures may vary considerably depending on the environmental conditions during testing.

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

    Directory of Open Access Journals (Sweden)

    Luis Segu

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-07-06

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

  18. Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

    DEFF Research Database (Denmark)

    Claesson, M H; Endel, B; Ulrik, J

    1994-01-01

    Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b...

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

    Science.gov (United States)

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

    2017-02-23

    Hawthorn (Crataegus pinnatifida Bunge) leave have been used to treat cardiovascular diseases in China and Europe. Hawthorn leave flavonoids (HLF) are the main part of extraction. Whether hawthorn leave flavonoids could attenuate the development of atherosclerosis and the possible mechanism remain unknown. High-fat diet (HFD) mixed with HLF at concentrations of 5mg/kg and 20mg/kg were administered to apolipoprotein E (apoE) knock out mice. 16 weeks later, mouse serum was collected to determine the lipid profile while the mouse aorta dissected was prepared to measure the lesion area. Hepatic mRNA of genes involved in lipid metabolism were determined. Peritoneal macrophages were collected to study the impact of HLF on cholesterol efflux, formation of foam cell and the expression of ATP binding cassette transporter A1 (ABCA1). Besides, in vivo reverse cholesterol transport (RCT) was conducted. HLF attenuated the development of atherosclerosis that the mean atherosclerotic lesion area in en face aortas was reduced by 23.1% (Pflavonoids can slow down the development of atherosclerosis in apoE knockout mice via induced expression of genes involved in antioxidant activities, inhibition of the foam cell formation and promotion of RCT in vivo, which implies the potential use in the prevention of atherosclerosis. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

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

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    Natalia Madrid-López

    2017-10-01

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

  1. Knock-out reactions

    International Nuclear Information System (INIS)

    de Forest, T. Jr.

    1977-01-01

    It is pointed out that the primary motivation for performing high energy single nucleon knock-out reactions is based on the concept of quasi-elastic scattering. The validity of and corrections to the partial wave impulse approximation and kinematical invariance of knock-out reactions and tests of the reaction mechanism are treated. The effect of distortions on the momentum distribution in the effective momentum approximation for given parameters are plotted. 12 references

  2. Role of interferon-gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-gamma knock-out mice

    DEFF Research Database (Denmark)

    Nansen, A; Christensen, Jan Pravsgaard; Röpke, C

    1998-01-01

    , a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN-gamma knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may...

  3. Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice.

    Science.gov (United States)

    Gruss, Michael; Braun, Katharina

    2004-07-01

    The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.

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

    Science.gov (United States)

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

    2017-05-01

    Regulatory T cells (T regs ) have been recognized as central mediators for maintaining peripheral tolerance and limiting autoimmune diseases. The loss of T regs or their function has been associated with exacerbation of autoimmune disease. However, the temporary loss of T regs in the chronic spontaneous disease model has not been investigated. In this study, we evaluated the role of T regs in a novel chronic spontaneous glomerulonephritis model of B cell lymphoma 2-interacting mediator (Bim) knock-out mice by transient depleting T regs . Bim is a pro-apoptotic member of the B cell lymphoma 2 (Bcl-2) family. Bim knock-out (Bim -/- ) mice fail to delete autoreactive T cells in thymus, leading to chronic spontaneous autoimmune kidney disease. We found that T reg depletion in Bim -/- mice exacerbated the kidney injury with increased proteinuria, impaired kidney function, weight loss and greater histological injury compared with wild-type mice. There was a significant increase in interstitial infiltrate of inflammatory cells, antibody deposition and tubular damage. Furthermore, the serum levels of cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17α, interferon (IFN)-γ and tumour necrosis factor (TNF)-α were increased significantly after T reg depletion in Bim -/- mice. This study demonstrates that transient depletion of T regs leads to enhanced self-reactive T effector cell function followed by exacerbation of kidney disease in the chronic spontaneous kidney disease model of Bim-deficient mice. © 2017 British Society for Immunology.

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

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    Portugal L.R.

    2006-01-01

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

  6. Knocking out or pharmaceutical inhibition of fatty acid binding protein 4 (FABP4) alleviates osteoarthritis induced by high-fat diet in mice.

    Science.gov (United States)

    Zhang, C; Chiu, K Y; Chan, B P M; Li, T; Wen, C; Xu, A; Yan, C H

    2018-06-01

    Adipokines play roles in the pathogenesis of osteoarthritis (OA). Fatty acid binding protein 4 (FABP4) is a novel adipokine that is closely associated with obesity and metabolic diseases. The aim of this study was to discover the potential role of FABP4 in OA. Seventy-two FABP4 knockout mice (KO) in C57BL/6N background and wild-type littermates (WT) (male, 6-week-old) were fed with a high-fat diet (HFD, 60% calorie) or standard diet (STD, 11.6% calorie) for 3 months, 6 months and 9 months (n = 6 each). In the parallel study, forty-eight 6-week-old male WT mice were fed with HFD or STD, and simultaneously treated with daily oral gavage of selective FABP4 inhibitor BMS309403 (15 mg/kg/d) or vehicle for 4 months and 6 months (n = 6 each). Serum FABP4 and cartilage oligomeric matrix protein (COMP) concentration was quantified. Histological assessment of knee OA and micro-CT analysis of subchondral bone were performed. HFD induced obesity in mice. After 3 months and 6 months of HFD, KO mice showed alleviated cartilage degradation and synovitis, with significantly lower COMP, modified Mankin OA score, and MMP-13/ADAMTS4 expression. After 6 months and 9 months of HFD, KO mice showed less osteophyte formation and subchondral bone sclerosis. Chronic treatment of BMS309403 for 4 months and 6 months significantly alleviated cartilage degradation, but had no effects on the subchondral bone. Knocking out or pharmaceutical inhibition of FABP4 did not have significant effects on lean mice fed with STD. Knocking out or pharmaceutical inhibition of FABP4 alleviates OA induced by HFD in mice. Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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

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

    2016-10-01

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

  8. Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

    Science.gov (United States)

    Horio, Mao; Ishima, Tamaki; Fujita, Yuko; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji

    2013-05-01

    d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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    Guoyong Yin

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

  10. Podocyte specific knock out of selenoproteins does not enhance nephropathy in streptozotocin diabetic C57BL/6 mice

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    Carlson Bradley A

    2008-07-01

    Full Text Available Abstract Background Selenoproteins contain selenocysteine (Sec, commonly considered the 21st genetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/or through their roles in the maintenance of intracellular redox balance. Since oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteins protect against this complication of diabetes. Methods C57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins (denoted in this paper as PodoTrsp-/- and control mice were made diabetic by intraperitoneal injection of streptozotocin, or were injected with vehicle. Blood glucose, body weight, microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers of oxidative stress were assessed. Results After 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of blood glucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff staining to examine mesangial matrix expansion also demonstrated no difference between control and PodoTrsp-/- mice after 6 months of diabetes, and there were no differences in immunohistochemical stainings for nitrotyrosine or NAD(PH dehydrogenase, quinone 1. Conclusion Loss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does not lead to increased oxidative stress as assessed by nitrotyrosine and NAD(PH dehydrogenase, quinone 1 immunostaining, nor does it lead to worsening nephropathy.

  11. Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Jauhiainen, Matti; Moser, Markus

    2008-01-01

    To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fol...

  12. 2-Methyl-6-(phenylethynyl pyridine (MPEP reverses maze learning and PSD-95 deficits in Fmr1 knock-out mice.

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    Réno Michelle Gandhi

    2014-03-01

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

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

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    Rita Delgado Marques

    2013-10-01

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

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

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

    2017-01-01

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

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

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    Pascal Jorratt

    2017-11-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

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    Susanna Pietropaolo

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

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

    Science.gov (United States)

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

    2017-07-07

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

  19. Staggering but not knocked out

    Energy Technology Data Exchange (ETDEWEB)

    Anon.

    2012-11-01

    Italy's PV market is staggering like a boxer almost knocked out. It has been hit hard by the country's deep economic recession. Conto Energia V has been yet another blow with cuts of up to 40 % in the solar feed-in tariffs. But the situation is not hopeless.

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

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    Kelsey Moore

    2016-11-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Karine eSonzogni-Desautels

    2015-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kasprzak, Kazimierz S. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Diwan, Bhalchandra A. [Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Kaczmarek, Monika Z. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Logsdon, Daniel L. [Laboratory Animal Sciences Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Fivash, Mathew J. [Data Management Services, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Salnikow, Konstantin, E-mail: salnikok@mail.nih.gov [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States)

    2011-11-15

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  5. Creation of knock out and knock in mice by CRISPR/Cas9 to validate candidate genes for human male infertility, interest, difficulties and feasibility.

    Science.gov (United States)

    Kherraf, Zine-Eddine; Conne, Beatrice; Amiri-Yekta, Amir; Kent, Marie Christou; Coutton, Charles; Escoffier, Jessica; Nef, Serge; Arnoult, Christophe; Ray, Pierre F

    2018-06-15

    High throughput sequencing (HTS) and CRISPR/Cas9 are two recent technologies that are currently revolutionizing biological and clinical research. Both techniques are complementary as HTS permits to identify new genetic variants and genes involved in various pathologies and CRISPR/Cas9 permits to create animals or cell models to validate the effect of the identified variants, to characterize the pathogeny of the identified variants and the function of the genes of interest and ultimately to provide ways of correcting the molecular defects. We analyzed a cohort of 78 infertile men presenting with multiple morphological anomalies of the sperm flagella (MMAF), a severe form of male infertility. Using whole exome sequencing (WES), homozygous mutations in autosomal candidate genes were identified in 63% of the tested subjects. We decided to produce by CRISPR/cas9 four knock-out (KO) and one knock-in (KI) mouse lines to confirm these results and to increase our understanding of the physiopathology associated with these genetic variations. Overall 31% of the live pups obtained presented a mutational event in one of the targeted regions. All identified events were insertions or deletions localized near the PAM sequence. Surprisingly we observed a high rate of germline mosaicism as 30% of the F1 displayed a different mutation than the parental event characterized on somatic tissue (tail), indicating that CRISPR/Cas9 mutational events kept happening several cell divisions after the injection. Overall, we created mouse models for 5 distinct loci and in each case homozygous animals could be obtained in approximately 6 months. These results demonstrate that the combined use of WES and CRISPR/Cas9 is an efficient and timely strategy to identify and validate mutations responsible for infertility phenotypes in human. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ken Murakami

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

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

    Science.gov (United States)

    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.

  8. Susceptibility of IFN? or IL-12 knock-out and SCID mice to infection with two microsporidian species, Encephalitozoon cuniculi and E. intestinalis

    Czech Academy of Sciences Publication Activity Database

    Salát, Jiří; Sak, Bohumil; Le, T.; Kopecký, Jan

    2004-01-01

    Roč. 51, č. 4 (2004), s. 275-282 ISSN 0015-5683 R&D Projects: GA AV ČR IAA6022101; GA ČR GP524/03/D167 Grant - others:Bravo! Program(US) MIRT T37TW00036-01 Institutional research plan: CEZ:AV0Z6022909 Keywords : Microsporidia * Encephalitozoon * mice Subject RIV: EC - Immunology Impact factor: 0.837, year: 2004

  9. Proton knock-out in Hall A

    International Nuclear Information System (INIS)

    Jager, K. de

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ellen R Busby

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

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

    Directory of Open Access Journals (Sweden)

    Alain M Gardier

    2013-08-01

    Full Text Available Why antidepressants vary in terms of efficacy is currently unclear. Despite the leadership of Selective serotonin reuptake inhibitors (SSRIs in the treatment of depression, the precise neurobiological mechanisms involved in their therapeutic action are poorly understood. A better knowledge of molecular interactions between monoaminergic system, pre- and post-synaptic partners, brain neuronal circuits and regions involved may help to overcome limitations of current treatments and to identify new therapeutic targets. Intracerebral in vivo microdialysis (ICM already provided important information about the brain mechanism of action of antidepressants first in anesthetized rats in the early 90s, and since then in conscious wild-type or knockout mice. The principle of ICM is based on the balance between release of neurotransmitters (e.g., monoamines, and re-uptake by selective transporters (e.g., SERT for serotonin 5-HT. Complementary to electrophysiology, this technique reflects presynaptic monoamines release and intrasynaptic events corresponding to ≈ 80% of whole brain tissue content. The inhibitory role of serotonergic autoreceptors infers that they limit somatodendritic and nerve terminal 5-HT release. It has been proposed that activation of 5-HT1A and 5-HT1B receptor sub-types limit the antidepressant-like activity of Selective Serotonin Reuptake Inhibitors (SSRI. This hypothesis is based partially on results obtained in ICM experiments performed in naïve, non-stressed Rodents. The present review will first remind the principle and methodology of ICM performed in mice. The crucial need of developing animal models that display anxiety and depression-like behaviors, neurochemical and brain morphological phenotypes reminiscent of these mood disorders in Human, will be underlined. Recently developed genetic mouse models have been generated to independently manipulate 5-HT1A auto and hetero-receptors and ICM helped to clarify the role of the

  12. Knock out for subthreshold pion production

    International Nuclear Information System (INIS)

    Guet, C.; Prakash, M.

    1984-05-01

    The contribution of nucleon-nucleon-single collisions to subthreshold pion production in hadron-nucleus and nucleus-nucleus collisions, Esub(Lab) < 300 A MeV is investigated within a knock-out type model. This contribution might be important for energies higher than about 150 MeV/nucleon but decrease strongly with decreasing beam energy

  13. Parallel knock-out schemes in networks

    NARCIS (Netherlands)

    Broersma, H.J.; Fomin, F.V.; Woeginger, G.J.

    2004-01-01

    We consider parallel knock-out schemes, a procedure on graphs introduced by Lampert and Slater in 1997 in which each vertex eliminates exactly one of its neighbors in each round. We are considering cases in which after a finite number of rounds, where the minimimum number is called the parallel

  14. Pion-induced knock-out reactions

    International Nuclear Information System (INIS)

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

    1977-01-01

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

  15. Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP(-/-) Mice.

    Science.gov (United States)

    Fan, Jing; Stemkowski, Patrick L; Gandini, Maria A; Black, Stefanie A; Zhang, Zizhen; Souza, Ivana A; Chen, Lina; Zamponi, Gerald W

    2016-01-01

    Genetic ablation of cellular prion protein (PrP(C)) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrP(C) profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrP(C). The amplitude of voltage sag, a characteristic of activating HCN channel current (I h), was decreased in null mice. Moreover, I h peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrP(C). These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability.

  16. Eliminating graphs by means of parallel knock-out schemes

    NARCIS (Netherlands)

    Broersma, H.J.; Fomin, F.V.; Královic, R.; Woeginger, G.J.

    2007-01-01

    In 1997 Lampert and Slater introduced parallel knock-out schemes, an iterative process on graphs that goes through several rounds. In each round of this process, every vertex eliminates exactly one of its neighbors. The parallel knock-out number of a graph is the minimum number of rounds after which

  17. Eliminating graphs by means of parallel knock-out schemes

    NARCIS (Netherlands)

    Broersma, Haitze J.; Fomin, F.V.; Královič, R.; Woeginger, Gerhard

    In 1997 Lampert and Slater introduced parallel knock-out schemes, an iterative process on graphs that goes through several rounds. In each round of this process, every vertex eliminates exactly one of its neighbors. The parallel knock-out number of a graph is the minimum number of rounds after which

  18. Surface sensitivity of nuclear-knock-out form factors

    International Nuclear Information System (INIS)

    Fratamico, G.

    1984-01-01

    A numerical calculation has been performed to investigate the sensitivity of nuclear-knock-out form factors to nuclear-surface behaviour of bound-state wave functions. The result of our investigation suggests that one can extract the bound-state behaviour at the surface from experimental information on nuclear-knock-out form factors

  19. Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

    Science.gov (United States)

    Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

    2015-01-21

    Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. Copyright © 2015 the authors 0270-6474/15/350943-17$15.00/0.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF(2L/2LCk-cre)). A severe impairment...... specific for the serotonin 2A receptor (5-HT(2A)R) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT(2A)Rs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered...... was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT(2A) and 5-HT(1A) mRNA expression but normal 5-HT(2C) content in these brain regions in BDNF(2L/2LCk-cre) mice. We investigated whether the reduction in frontal 5-HT(2A...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF(2L/2LCk-cre)). A severe impairmen...

  2. Single proton knock-out from 24F

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  3. Caracterización del 'Knock out' en Boxeo

    OpenAIRE

    Pic-Aguilar, Miguel; Sánchez-López, Carmen R.; Blanco Villaseñor, Ángel

    2016-01-01

    El objetivo de este trabajo es identificar las cuatro últimas acciones motrices emitidas (golpes) por boxeadores campeones del mundo de los pesos pesados y así poder caracterizar el 'Knock out' en boxeo. Para ello, hemos desarrollado una herramienta de observación que consta de cuatro criterios y 35 categorías. Para la selección de la muestra se tuvo en cuenta dos requisitos: haberse proclamado campeón del mundo del peso pesado durante el período que comprende 1921-2007 (desde Jack Dempsey ha...

  4. Theoretical analysis of knock-out release of fission products from nuclear fuels

    International Nuclear Information System (INIS)

    Yamagishi, S.

    1975-01-01

    The knock-out release of fission products is studied theoretically. The general equations of knock-out release are derived, assuming that a fission fragment passing through the surface of nuclear fuels knocks out a local region of the surface with an effective thickness and an effective cross-sectional area. Using these equations, the knock-out release of fission gases is calculated for various cases. The conditions under which the knock-out coefficients (the average number of uranium atoms knocked out by one fission fragment) is obtainable are clarified by experiments on the knock-out release of fission gases. A method of determining the effective thickness and the effective cross-sectional area of a knock-out region is proposed. (Auth.)

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

    Science.gov (United States)

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

    2017-07-01

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

  6. PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice

    NARCIS (Netherlands)

    McBride, P. A.; Eikelenboom, P.; Kraal, G.; Fraser, H.; Bruce, M. E.

    1992-01-01

    Abnormal forms of a host protein, PrP, accumulate in the central nervous system in scrapie-affected animals. Here, PrP protein was detected immunocytochemically in tissue sections of spleen, lymph node, Peyer's patches, thymus, and pancreas from uninfected mice and from mice infected with a range of

  7. Computer simulation of the spatial distribution of optical radiation arising from knocked-out excited particles

    International Nuclear Information System (INIS)

    Gokov, S.P.; Gritsyna, V.V.; Koval', A.G.; Kovtunenko, Yu.I.; Shevchenko, D.I.

    2004-01-01

    The new approach for the explanation of the spatial distribution of the optical radiation arising from knocked-out excited particles is given. Calculated and experimental data for Al (λ=396.1 nm) and Mg (λ=383.8 nm) knocked-out by Ar + (20 keV) beam from MgAl 2 O 4 surface are compared [ru

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

    Science.gov (United States)

    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.

  9. On a calculation of nucleon knock-out cross sections in a collision of relativistic nuclei

    International Nuclear Information System (INIS)

    Goryachev, B.I.; Lin'kova, N.V.

    1985-01-01

    It is shown that in the framework of the two-stage model one can obtain knock-out cross sections of the given number of nucleons from the nucleus-target at a certain number of nucleons knocked out from the nucleus-projectile. The first stage is considered as a fast process of nucleon collisions of interacting nuclei which is completed with knock out of one or several nucleons. The second stage-comparatively slow - is related to de-excitation of nuclei-fragments

  10. On the knock-out mechanism for the 12C(P,α)9B reaction

    International Nuclear Information System (INIS)

    Hassan, M.Y.M.; Ismail, E.H.; Rabie, A.

    1978-01-01

    The mechanism of the reaction 12 C(P,α) 9 B is studied using zero range distorted wave Born approximation. The knock out mechanism is assumed to represent this reaction both in the forward and backward angles. (orig.) [de

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

    Science.gov (United States)

    Wilson, Bradley

    1998-01-01

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

  12. Temperature dependence of knocking-out cross sections of a bound atom from the lattice site

    International Nuclear Information System (INIS)

    Zhdanov, S.K.; Pletnev, V.V.

    1981-01-01

    The total cross section of atom knocking-out from the lattice site is calculated with the atom binding in the lattice site taken into account. The intermediate case of atom being preads over the bottom of a spherical potential well is considered (the case of intermediate temperatures). Thus the target temperature parameter enters the equation for the total cross section of atom knocking-out

  13. Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

    Science.gov (United States)

    Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg; Kretzschmar, Hans; Westaway, David

    2017-01-01

    Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

  14. High-temperature expansion and knock-out properties of moulding sands with water glass

    Directory of Open Access Journals (Sweden)

    Major-Gabryś K.

    2007-01-01

    Full Text Available The article focuses on the topic of improving the knock-out properties of moulding sand with water glass and ester hardener. It is settled that the cause of worse knock-out properties of moulding sand can be brought by their thermal expansion in increased temperatures. There is a presentation of the influence of different additives, containing Al2O3, on moulding sands’ expansion in increased temperatures. Within the frames of research, there was an elaboration of the influence of authors own additive- Glassex, on the expansion phenomenon of moulding sands with water glass and ester hardener. It is concluded, that the new additive stops the expansion of moulding sands and as well it improves their knock-out properties.

  15. A model of knock-out of oxygen by charged particle irradiation of Bi-2212

    International Nuclear Information System (INIS)

    Bandyopadhyay, S.K.; Sen, Pintu; Barat, P.; Mukherjee, P.; Das, S.K.; Ghosh, B.

    1996-01-01

    A model of knock-out of oxygen by charged particle (α and proton) irradiation of Bi 2 Sr 2 CaCu 2 O 8+x (Bi-2212) is proposed on the basis of Monte Carlo TRIM calculations. In Bi-2212, the loosely bound excess oxygen is vulnerable to be displaced by particle irradiation. Binding energy and hence, displacement energy of this loosely bound excess oxygen is less compared to that of stoichiometric lattice bound oxygen and other atoms. The displaced or knocked out oxygen goes to pores or intergranular region and generates large pressure inside the sample. Because of porosity of the material, this displaced oxygen diffuses out and there is a net reduction of oxygen content of the sample. The irradiation induced oxygen knock-out is dominant in the bulk where nonionizing energy loss is maximum. (author). 29 refs., 1 fig., 3 tabs

  16. On the interpretation of (e,e'p) knock-out reaction

    International Nuclear Information System (INIS)

    Dieperink, A.E.L.

    The basic physics in (e,e'p) knock-out reactions is illustrated assuming that the knocked-out proton can be treated as a plane wave (PWIA). Corrections for distortion and absorption of the outgoing proton can, in principle, be calculated to a good approximation with an optical potential. The spectral function is characterized in terms of its energy moments, the lowest of which can be incorporated in an independent particle shell model (IPSM): occupatiomn probability (zeroth moment) and the mean removal energy (centroid energy). Deviations from IPSM are discussed: binding energy sum rule, A=3 nuclei, 6 Li, and fragmentation of single-particle strength

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

    Science.gov (United States)

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    unintuitive and difficult to determine. In this article, we propose a novel MVPA method for group analysis of high-dimensional data that overcomes the drawbacks of the current techniques. Our approach explicitly aims to identify all relevant variations using a "knock-out" strategy and the Random Forest...

  19. Downy mildew of Double Knock Out® rose caused by Peronospora sparsa in Maryland

    Science.gov (United States)

    Roses are one of the most popular and economically important ornamental plants worldwide. In the last 17 years, Knock Out® roses (Rosa x 'Radtko') have been widely used in public and private gardens across the U.S. due to their disease resistance, self-cleaning, drought tolerance and multiple-bloomi...

  20. Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

    Science.gov (United States)

    Sangaletti, Rachele; D'Amico, Massimo; Grant, Jeff; Della-Morte, David; Bianchi, Laura

    2017-08-01

    Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

  1. Inflammation in Lafora Disease: Evolution with Disease Progression in Laforin and Malin Knock-out Mouse Models.

    Science.gov (United States)

    López-González, Irene; Viana, Rosa; Sanz, Pascual; Ferrer, Isidre

    2017-07-01

    Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a -/- (laforin knock-out) and Epm2b -/- (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 messenger RNAs (mRNAs) are significantly increased in Epm2a -/- mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα, and Il10ra mRNAs are significantly upregulated in Epm2b -/- at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα, and Cox2 particularly in Epm2b -/- mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b -/- mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD.

  2. Post-irradiation studies on knock-out and pseudo-recoil releases of fission products from fissioning UO2

    International Nuclear Information System (INIS)

    Yamagishi, S.; Tanifuji, T.

    1976-01-01

    By using post-irradiation techniques, in-pile releases of 133 Xe, sup(85m)Kr, 88 Kr, 87 Kr and 138 Xe from UO 2 fissioning at low temperatures below about 200 0 C are studied: these are analyzed into a time-dependent knock-out and time-independent pseudo-recoil releases. For the latter, a 'self knock-out' mechanism is proposed: when a fission fragment loses thoroughly its energy near the UO 2 surface and stops there, it will knock out the surface substances and accordingly the fragment (i.e. the fission product) will be released. The effective thickness of the layer where the self knock-out occurs is found to be approximately 7A. As for the knock-out release, the following is estimated from its dependence on various factors: the knock-out release of fission products occurs from the surface layer with the effective thickness of approximately 20A: the shape of UO 2 matrix knocked out by one fission fragment passing through the surface is equivalent to a cylinder approximately 32A diameter by approximately 27A thick, (i.e. the knock-out coefficient for UO 2 is approximately 660 uranium atoms per knock-out event). On the basis of the above estimations, the conclusions derived from the past in-pile studies of fission gas releases are evaluated. (Auth.)

  3. Quasielastic knock out of light fragments from 12C and 16O by intermediate energy pions

    International Nuclear Information System (INIS)

    Abramov, B.M.; Borodin, Yu.A.; Bulychev, S.A.

    2006-01-01

    Using 0.72 GeV s -1 pulse π - -meson beam one studied the quasi-elastic knocking out of deuterons and of tritons from 12 C and 16 O nuclei. One derived the quasi-deuteron intranuclear motion pulse distributions, the residual nucleus excitation energy spectra and the effective number of quasi-deuterons. The parameters of quasi-deuteron intranuclear motion pulse distributions are in line with the measurement results for other beams. The effective numbers of quasi-deuterons in nuclei from 6 Li up to 16 O do not depend on the atomic number. One observed knocking out of tritons from the mentioned nuclei enabling to evaluate the cross section of elastic pion-triton backscattering [ru

  4. 13C(α,n)16O reaction as the knock-out exchange process

    International Nuclear Information System (INIS)

    Kim, G.; Khajdarov, R.R.; Zaparov, Eh.A.

    2000-01-01

    S-factor for the 13 C(α,n) 16 O reaction is studied. In the framework of the simple phenomenological model this reaction is analysed as neutron knocked-out by α-particle exchange process. The analysis demonstrates the importance of taking into account 2p-state in 13 C. The 13 C(α,n) 16 O cross section is considered both as the knock-out exchange process and as it's combination with process through a compound nucleus. It was shown that for E α s value extrapolated to low energies is found to be noticeably larger that of R-matrix analysis. Different ways of improving the proposed model are discussed. (author)

  5. Histone deacetylase 6 inhibition reduces cysts by decreasing cAMP and Ca2+ in knock-out mouse models of polycystic kidney disease.

    Science.gov (United States)

    Yanda, Murali K; Liu, Qiangni; Cebotaru, Valeriu; Guggino, William B; Cebotaru, Liudmila

    2017-10-27

    Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 ( pkd1 ), primarily a signaling molecule, and PC2 ( pkd2 ), a Ca 2+ channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca 2+ was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca 2+ and increased ATP-simulated Ca 2+ release. HDAC6 inhibition reduced the release of Ca 2+ from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ -ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca 2+ levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. PrP0\\0 mice show behavioral abnormalities that suggest PrPC has a role in maintaining the cytoskeleton.

    Science.gov (United States)

    Background/Introduction. PrPC is highly conserved among mammals, but its natural function is unclear. Prnp ablated mice (PrP0/0) appear to develop normally and are able to reproduce. These observations seem to indicate that the gene is not essential for viability, in spite of it being highly conse...

  7. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhou

    Full Text Available Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG. In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1 and 0% (10 ng/μL sg2 and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group and 28.6% (50 ng/μL sg2+sg3 group. Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01 decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05. As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4, and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

  8. ANTXR2 Knock-Out Does Not Result in the Development of Hypertension in Rats.

    Science.gov (United States)

    Liu, Xiaoyan; Yuan, Wen; Li, Jing; Yang, Lei; Cai, Jun

    2017-02-01

    Our recent genetic study as well as robust evidences reported by previous genome-wide association studies (GWASs) have indicated that the single nucleotide polymorphism rs16998073, located near gene anthrax toxin receptor 2 (ANTXR2), was significantly associated with hypertension in Asians and Europeans. The aim of the present study was to determine whether ANTXR2 is the causal gene of hypertension at the 4q21 locus using an ANTXR2 knock-out model. Relative expression of ANTXR2 in Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were determined by real-time quantitative polymerase chain reaction and western blot analysis. ANTXR2 knock-out rats were created using CRISPR/Cas9-mediated genome editing and blood pressure values were measured in ANTXR2 -/- and wild type (WT) rats by tail-cuff method and carotid arterial catheterization method. Neither the mRNA nor protein levels of ANTXR2 were significantly different between tissues from SHRs and WKYs. To create ANTXR2 -/- rats, 67 base pairs were deleted in exon 1 of ANTXR2 using CRISPR/Cas9-mediated genome editing. ANTXR2 protein decreased significantly in aortas of ANTXR2 -/- rats, suggesting sufficient efficiency of ANTXR2 knock-out in this model. However, ANTXR2 -/- rats exhibited nearly the same blood pressure as WT rats at baseline conditions as well as during Angiotensin II (400ng/kg/min) infusion or high-salt diet treatment. These findings suggest that ANTXR2 might not be associated with hypertension and thus further functional analysis is warranted to identify the causal gene at this locus. © American Journal of Hypertension, Ltd 2016. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity.

    Science.gov (United States)

    Cingaram, Pradeep Kumar Reddy; Nyeste, Antal; Dondapati, Divya Teja; Fodor, Elfrieda; Welker, Ervin

    2015-01-01

    The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

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

    Science.gov (United States)

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

    2012-12-15

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

  11. Specific features of energy and spatial distribution of primary knocked-out atoms in monocrystals

    International Nuclear Information System (INIS)

    Taratin, A.M.; Vorob'ev, S.A.

    1978-01-01

    By simulation trajectories of 0.2 MeV protons in 1 μm thick Al monocrystal, the energy and spatial distributions of primary atoms knocked out by the protons (PKA) have been studied. Different orientations of the incident beam axis relative to the densely packed direction in the case of ''quasichanneling'' and ''chaotic'' scattering of particles by the crystal have been researched. The depth dependence of the number of generated PKA, their distribution in the plane transverse to the preferred direction, and the energy spectrum of PKA have been obtained. It is shown that the PKA volume density is higher than that obtained using evaluations not accounting for the crystalline structure, and the energy spectrum contains more low energy PKAs. A concept of the cross section of the PKA production on an atomic chain is introduced for ipterpretation of the data obtained

  12. Overlap knock-out effects in the CERN intersecting storage rings (ISR)

    CERN Document Server

    Gourber, J P; Myers, S

    1977-01-01

    Overlap knock-out arises from an overlap between frequencies present in a bunched beam and the betatron frequencies in a stack. The 'single ring' effect in the interaction of a bunched beam with a stack in the same ring. Here the coupling forces are fairly linear and are transmitted by machine elements. The 'two-ring' effect is the interaction of a bunched beam with a stack in the other ring. Here the coupling forces are nonlinear since they are produced by the beam-beam interaction. A brief outline of the general theory of these effects is given. The single ring and two-ring dipole effects have been observed and shown to cause a large increase in the transverse size of the stacked beam. (4 refs).

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

    Science.gov (United States)

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

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

  14. Mass spectrum of secondary ions knocked-out from copper surface by argon ion beam

    International Nuclear Information System (INIS)

    Koval', A.G.; Bobkov, V.V.; Klimovskij, Yu.A.; Fogel', Ya.M.

    1976-01-01

    The mass-spectrum of secondary ions was studied within a mass range of 1-400. The ions were knocked-out by the beam of ions Ar + from the copper surface with different content of oxygen and sulphur solved in the volume. The studies were conducted at three temperatures of the target. The atomic and molecular ions of the metal matrix, volumetric impurities of metal and ions of chemical compounds molecules of the metal under study with gas particles adsorbed on its surface and atoms of the metal volumetric admixtures may be observed in the mass spectrum. Detection of secondary ions of the copper multi-atomic complexes and ions of these complexes compounds with the adsorbed molecules is of interest

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

  16. Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice

    NARCIS (Netherlands)

    Kuiper, Esther J.; Roestenberg, Peggy; Ehlken, Christoph; Lambert, Vincent; van Treslong-de Groot, Henny Bloys; Lyons, Karen M.; Agostini, Hans-Jürgen T.; Rakic, Jean-Marie; Klaassen, Ingeborg; van Noorden, Cornelis J. F.; Goldschmeding, Roel; Schlingemann, Reinier O.

    2007-01-01

    Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Endres, Thomas; Lessmann, Volkmar

    2012-01-01

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

  19. Characterization of glutamate carboxypeptidase II knock-out mice generated by TALEN technology

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, Petr; Šácha, Pavel; Sedláček, Radislav; Konvalinka, Jan

    2017-01-01

    Roč. 15, č. 1 (2017), s. 44 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : glutamate carboxypeptidase II * TALEN technology Subject RIV: CE - Biochemistry

  20. Transmissibility of H-Type Bovine Spongiform Encephalopathy to Hamster PrP Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Okada

    Full Text Available Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE can be distinguished from classical (C- BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrPSc. H-BSE is transmissible to wild-type mice-with infected mice showing a long survival period that is close to their normal lifespan-but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrPSc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrPSc (PrPres in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrPSc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrPres in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrPres in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.

  1. Peroxiredoxin 6 promotes upregulation of the prion protein (PrP in neuronal cells of prion-infected mice

    Directory of Open Access Journals (Sweden)

    Wagner Wibke

    2012-12-01

    Full Text Available Abstract Background It has been widely established that the conversion of the cellular prion protein (PrPC into its abnormal isoform (PrPSc is responsible for the development of transmissible spongiform encephalopathies (TSEs. However, the knowledge of the detailed molecular mechanisms and direct functional consequences within the cell is rare. In this study, we aimed at the identification of deregulated proteins which might be involved in prion pathogenesis. Findings Apolipoprotein E and peroxiredoxin 6 (PRDX6 were identified as upregulated proteins in brains of scrapie-infected mice and cultured neuronal cell lines. Downregulation of PrP gene expression using specific siRNA did not result in a decrease of PRDX6 amounts. Interestingly, selective siRNA targeting PRDX6 or overexpression of PRDX6 controlled PrPC and PrPSc protein amounts in neuronal cells. Conclusions Besides its possible function as a novel marker protein in the diagnosis of TSEs, PDRX6 represents an attractive target molecule in putative pharmacological intervention strategies in the future.

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

    Science.gov (United States)

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

    2015-01-01

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

  3. A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

    Science.gov (United States)

    Bouhy, Delphine; Juneja, Manisha; Katona, Istvan; Holmgren, Anne; Asselbergh, Bob; De Winter, Vicky; Hochepied, Tino; Goossens, Steven; Haigh, Jody J; Libert, Claude; Ceuterick-de Groote, Chantal; Irobi, Joy; Weis, Joachim; Timmerman, Vincent

    2018-01-01

    Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces

  4. Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions.

    Science.gov (United States)

    Chapuis, Jérôme; Moudjou, Mohammed; Reine, Fabienne; Herzog, Laetitia; Jaumain, Emilie; Chapuis, Céline; Quadrio, Isabelle; Boulliat, Jacques; Perret-Liaudet, Armand; Dron, Michel; Laude, Hubert; Rezaei, Human; Béringue, Vincent

    2016-02-05

    Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrP(Sc), a misfolded isoform of the host-encoded prion protein PrP(C). Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrP(Sc) in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to 'mutate' conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrP(Sc)), to transgenic mice overexpressing either human or the VRQ allele of ovine PrP(C). In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrP(Sc) biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the 'physical' cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells. Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of

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

    Science.gov (United States)

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

  6. Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

    Science.gov (United States)

    Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

    2018-02-01

    Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

    OpenAIRE

    Sakudo, Akikazu; Onodera, Takashi

    2015-01-01

    Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout (Prnp ?/?) mice. So far, six types of Prnp ?/? mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not ...

  8. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig, E-mail: blackstc@ninds.nih.gov

    2016-11-15

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  9. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    International Nuclear Information System (INIS)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

    2016-01-01

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  10. Analysis of different multiplicities and their interference in quasi-elastic cluster knock-out by fast hadrons

    International Nuclear Information System (INIS)

    Golovanova, N.F.; Ibraeva, E.T.; Neudatchin, V.G.

    1978-01-01

    Different multiplicities and their interference in hadron scattering have been investigated on the basis of a new dynamic approach to quasi-elastic knock-out of nucleon clusters by fast hadrons from light nuclei. It is shown that in the region of momentum transfer values p, where scattering multiplicities less than b are predominant, the effective numbers and form factors determined in Refs. 1) -- 3) no longer act as pure structural nuclear factors (b means the number of nucleons in the knocked-out cluster). These characteristics are significantly dependent on the process dynamics. Only in the region of values p, where the maximum hadron scattering multiplicity b is realized, the effective numbers and form factors do assume the purely structural meaning. (auth.)

  11. Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

    Science.gov (United States)

    Van Bogaert, Inge N A; Sabirova, Julia; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

    2009-06-01

    The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Hacking, Douglas F

    2008-12-01

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

  14. Rescue of Learning and Memory Deficits in the Human Nonsyndromic Intellectual Disability Cereblon Knock-Out Mouse Model by Targeting the AMP-Activated Protein Kinase-mTORC1 Translational Pathway.

    Science.gov (United States)

    Bavley, Charlotte C; Rice, Richard C; Fischer, Delaney K; Fakira, Amanda K; Byrne, Maureen; Kosovsky, Maria; Rizzo, Bryant K; Del Prete, Dolores; Alaedini, Armin; Morón, Jose A; Higgins, Joseph J; D'Adamio, Luciano; Rajadhyaksha, Anjali M

    2018-03-14

    A homozygous nonsense mutation in the cereblon ( CRBN ) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out ( Crbn KO ) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and Crbn KO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that Crbn KO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult Crbn KO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, Crbn KO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory. SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon ( CRBN ) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an

  15. Absence of Evidence for a Causal Link between Bovine Spongiform Encephalopathy Strain Variant L-BSE and Known Forms of Sporadic Creutzfeldt-Jakob Disease in Human PrP Transgenic Mice.

    Science.gov (United States)

    Jaumain, Emilie; Quadrio, Isabelle; Herzog, Laetitia; Reine, Fabienne; Rezaei, Human; Andréoletti, Olivier; Laude, Hubert; Perret-Liaudet, Armand; Haïk, Stéphane; Béringue, Vincent

    2016-12-01

    Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrP C ) and the infecting pathological PrP assemblies (PrP Sc ) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrP C and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrP Sc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD. Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy

    Directory of Open Access Journals (Sweden)

    Kohei Yasunaga

    2018-01-01

    Full Text Available Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to “impaired autophagy flux”. Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles.

  17. Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

    Science.gov (United States)

    Zhang, Zhanquan; Qin, Guozheng; Li, Boqiang; Tian, Shiping

    2014-06-01

    Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

  18. Possibility of investigation of pion degrees of freedom in atomic nuclei with the help of quasielastic pions knocking out by high energy electrons

    CERN Document Server

    Neudachin, V G; Sviridova, L L

    2002-01-01

    The attention is paid to the interesting possibilities of studying the pion degrees of freedom in the atomic nuclei by means of the quasielastic knocking out of pion (e, ep) from the nuclei by the electrons with the energy of several GeV. It appears, that the pulse distribution of the pions, knocked out from the separate nucleons and the nuclei, is in the whole differ essentially different

  19. PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

    Science.gov (United States)

    Marshall, Karen E; Hughson, Andrew; Vascellari, Sarah; Priola, Suzette A; Sakudo, Akikazu; Onodera, Takashi; Baron, Gerald S

    2017-01-15

    Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion. Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C

  20. Experimental Models of Inherited PrP Prion Diseases.

    Science.gov (United States)

    Watts, Joel C; Prusiner, Stanley B

    2017-11-01

    The inherited prion protein (PrP) prion disorders, which include familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia, constitute ∼10%-15% of all PrP prion disease cases in humans. Attempts to generate animal models of these disorders using transgenic mice expressing mutant PrP have produced variable results. Although many lines of mice develop spontaneous signs of neurological illness with accompanying prion disease-specific neuropathological changes, others do not. Furthermore, demonstrating the presence of protease-resistant PrP species and prion infectivity-two of the hallmarks of the PrP prion disorders-in the brains of spontaneously sick mice has proven particularly challenging. Here, we review the progress that has been made toward developing accurate mouse models of the inherited PrP prion disorders. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  1. Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

    Science.gov (United States)

    Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

    2017-01-01

    Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

  2. Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

    Science.gov (United States)

    Sakudo, Akikazu; Onodera, Takashi

    2015-01-01

    Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout (Prnp−/−) mice. So far, six types of Prnp−/− mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not fully reveal PrP functions. Recently, varieties of Prnp−/− cell lines established from Prnp−/− mice have contributed to the analysis of PrP functions. In this mini-review, we focus on Prnp−/− cell lines and summarize currently available Prnp−/− cell lines and their characterizations. In addition, we introduce the recent advances in the methodology of cell line generation with knockout or knockdown of the PrP gene. We also discuss how these cell lines have provided valuable insights into PrP functions and show future perspectives. PMID:25642423

  3. Expression of Id2 in the Second Heart Field and Cardiac Defects in Id2 Knock-Out Mice

    NARCIS (Netherlands)

    Jongbloed, M. R. M.; Vicente-Steijn, R.; Douglas, Y. L.; Wisse, L. J.; Mori, K.; Yokota, Y.; Bartelings, M. M.; Schalij, M. J.; Mahtab, E. A.; Poelmann, R. E.; Gittenberger-De Groot, A. C.

    2011-01-01

    The inhibitor of differentiation Id2 is expressed in mesoderm of the second heart field, which contributes myocardial and mesenchymal cells to the primary heart tube. The role of Id2 in cardiac development is insufficiently known. Heart development was studied in sequential developmental stages in

  4. Impaired Ventilatory and Thermoregulatory Responses to Hypoxic Stress in Newborn Phox2b Heterozygous Knock-Out Mice

    OpenAIRE

    Ramanantsoa, Nelina; Matrot, Boris; Vardon, Guy; Lajard, Anne-Marie; Voituron, Nicolas; Dauger, Stéphane; Denjean, André; Hilaire, Gérard; Gallego, Jorge

    2011-01-01

    The Phox2b genesis necessary for the development of the autonomic nervous system, and especially, of respiratory neuronal circuits. In the present study, we examined the role of Phox2b in ventilatory and thermoregulatory responses to hypoxic stress, which are closely related in the postnatal period. Hypoxic stress was generated by strong thermal stimulus, combined or not with reduced inspired O2. To this end, we exposed 6-day-old Phox2b +/? pups and their wild-type littermates (Phox2b +/+) to...

  5. Impaired Ventilatory and Thermoregulatory Responses to Hypoxic Stress in Newborn Phox2b Heterozygous Knock-Out Mice

    Science.gov (United States)

    Ramanantsoa, Nelina; Matrot, Boris; Vardon, Guy; Lajard, Anne-Marie; Voituron, Nicolas; Dauger, Stéphane; Denjean, André; Hilaire, Gérard; Gallego, Jorge

    2011-01-01

    The Phox2b genesis necessary for the development of the autonomic nervous system, and especially, of respiratory neuronal circuits. In the present study, we examined the role of Phox2b in ventilatory and thermoregulatory responses to hypoxic stress, which are closely related in the postnatal period. Hypoxic stress was generated by strong thermal stimulus, combined or not with reduced inspired O2. To this end, we exposed 6-day-old Phox2b+/− pups and their wild-type littermates (Phox2b+/+) to hypoxia (10% O2) or hypercapnia (8% CO2) under thermoneutral (33°C) or cold (26°C) conditions. We found that Phox2b+/− pups showed less normoxic ventilation (VE) in the cold than Phox2b+/+ pups. Phox2b+/− pups also showed lower oxygen consumption (VO2) in the cold, reflecting reduced thermogenesis and a lower body temperature. Furthermore, while the cold depressed ventilatory responses to hypoxia and hypercapnia in both genotype groups, this effect was less pronounced in Phox2b+/− pups. Finally, because serotonin (5-HT) neurons are pivotal to respiratory and thermoregulatory circuits and depend on Phox2b for their differentiation, we studied 5-HT metabolism using high pressure liquid chromatography, and found that it was altered in Phox2b+/− pups. We conclude that Phox2b haploinsufficiency alters the ability of newborns to cope with metabolic challenges, possibly due to 5-HT signaling impairments. PMID:21977017

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular nuc...

  7. A neuroanatomical and physiological study of the non-image forming visual system of the cone-rod homeobox gene (Crx) knock out mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Rath, Martin F; Lund-Andersen, Casper

    2010-01-01

    The anatomy and physiology of the non-image forming visual system was investigated in a visually blind cone-rod homeobox gene (Crx) knock-out mouse (Crx(-)(/)(-)), which lacks the outer segments of the photoreceptors. We show that the suprachiasmatic nuclei (SCN) in the Crx(-/-) mouse exhibit...... melanopsin neurons or the SCN may be necessary for a normal function of the non-image forming system of the mouse. However, a change in the SCN of the Crx(-/-) mouse might also explain the observed circadian differences between the knock out mouse and wild type mouse....

  8. Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays.

    Science.gov (United States)

    Bezrutczyk, Margaret; Hartwig, Thomas; Horschman, Marc; Char, Si Nian; Yang, Jinliang; Yang, Bing; Frommer, Wolf B; Sosso, Davide

    2018-04-01

    Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H + symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  9. Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1 Improves Recombinant Protein Expression in the HEK293 Cell Line

    Directory of Open Access Journals (Sweden)

    Laura Abaandou

    2018-06-01

    Full Text Available Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA and small interfering RNA (siRNA, to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1 gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293 cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

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

    Directory of Open Access Journals (Sweden)

    Matthieu M Vermeren

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

  11. Predicting the names of the best teams after the knock-out phase of a cricket series.

    Science.gov (United States)

    Lemmer, Hermanus Hofmeyr

    2014-01-01

    Cricket players' performances can best be judged after a large number of matches had been played. For test or one-day international (ODI) players, career data are normally used to calculate performance measures. These are normally good indicators of future performances, although various factors influence the performance of a player in a specific match. It is often necessary to judge players' performances based on a small number of scores, e.g. to identify the best players after a short series of matches. The challenge then is to use the best available criteria in order to assess performances as accurately and fairly as possible. In the present study the results of the knock-out phase of an International Cricket Council (ICC) World Cup ODI Series are used to predict the names of the best teams by means of a suitably formulated logistic regression model. Despite using very sparse data, the methods used are reasonably successful. It is also shown that if the same technique is applied to career ratings, very good results are obtained.

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

    Science.gov (United States)

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

    2015-12-01

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

  13. Neurodegeneration and unfolded-protein response in mice expressing a membrane-tethered flexible tail of PrP.

    Directory of Open Access Journals (Sweden)

    Paolo Dametto

    Full Text Available The cellular prion protein (PrPC consists of a flexible N-terminal tail (FT, aa 23-128 hinged to a membrane-anchored globular domain (GD, aa 129-231. Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi". Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.

  14. Enhanced genome editing tools for multi-gene deletion knock-out approaches using paired CRISPR sgRNAs in CHO cells

    DEFF Research Database (Denmark)

    Schmieder, Valerie; Bydlinski, Nina; Strasser, Richard

    2017-01-01

    (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by deleting the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. Fut8 also served as model...

  15. Hypothalamic neurosecretory and circadian vasopressinergic neuronal systems in the blind cone-rod homeobox knock out mouse (Crx(-/-) ) and the 129sv wild type mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Rath, Martin Fredensborg; Møller, Morten

    2013-01-01

    circadian AVP-rhythm. We have in this study of the brown 129sv mouse and the visual blind cone-rod homeobox gene knock out mouse (Crx(-/-) ) with degeneration of the retinal rods and cones, but a preserved non-image forming optic system, studied the temporal Avp-expression in both the neurosecretory...

  16. Excited states of virtual clusters in a nucleus and the processes of quasi-elastic cluster knock-out at high energies

    International Nuclear Information System (INIS)

    Golovanova, N.F.; Il'in, I.M.; Neudatchin, V.G.; Smirnov, Yu.F.; Tchuvil'sky, Yu.M.

    1976-01-01

    The quasi-elastic knock-out of nucleon clusters from nuclei by an incident high-energy hadron is considered within the framework of the Glauber-Sitenko multiple scattering theory. It is shown that the significant contribution to the cross section for the process comes not only from the hadron elastic scattering by a nonexcited virtual cluster but also from collisions with an excited virtual cluster, accompanied by de-excitation of this cluster. This necessitates modification of the usual theory of quasi-elastic cluster knock-out. First, the angular correlations of the knocked-out cluster and scattered hadron are no longer determined by the momentum distribution of the cluster in the nucleus. They are determined by another form factor F(q) which can be called the modified momentum distribution. Secondly, the meaning and values of the effective numbers of clusters Nsup(eff) have been changed. Thirdly, the characteristics of the processes depend not only on the modulus of momentum q, which the cluster had in the nucleus, but also on its direction relative to an incident beam. A method has been developed for the calculation of the fractional parentage coefficients, which are necessary for the calculation of the cluster knock-out from the p-shell nuclei. (Auth.)

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

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2013-03-01

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

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

    Directory of Open Access Journals (Sweden)

    M Crispo

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

  19. Shape and structure of N=Z ^64Ge; Electromagnetic transition rates from the application of the Recoil Distance Method to knock-out reactions.

    Science.gov (United States)

    Starosta, K.; Dewald, A.

    2007-04-01

    Transition rate measurements are reported for the 2^+1 and 2^+2 states in the N=Z nucleus ^64Ge. The measurement was done utilizing the Recoil Distance Method (RDM) and a unique combination of state of the art instruments at the National Superconducting Cyclotron Laboratory (NSCL). States of interest were populated via an intermediate energy single neutron knock-out reaction. RDM studies of knock-out and fragmentation reaction products hold the promise of reaching far from stability and providing lifetime information for intermediate-spin excited states in a wide range of exotic nuclei. The large-scale Shell Model calculations applying the recently developed GXPF1A interaction are in excellent agreement with the above results. Theoretical analysis suggests that ^64Ge is a collective γ-soft anharmonic vibrator.

  20. Investigating of the Knocking Out Properties of Moulding Sands with New Inorganic Binders Used for Castings of Non-ferrous Metal Alloys in Comparison with the Previously Used

    Directory of Open Access Journals (Sweden)

    I. Izdebska-Szanda

    2012-12-01

    Full Text Available The article presents the results of investigations, which make a fragment of the broad-scale studies carried out as a part of the projectPOIG.01.01.02-00-015/09 “Advanced materials and technologies”.One of the objectives of the introduction of new inorganic binders is to provide a good knocking out properties of moulding sands, whilemaintaining an appropriate level of strength properties.Therefore, a logical continuation of the previous studies were carried out the tests knocking out properties of moulding sands with newinorganic binders, including making moulds, pouring them by the chosen of non-ferrous metal alloys, knoking-out, and determining theknocking out work.The results of the study were related to the research results obtained by applying the moulding sand performed by existing technology.

  1. Effect of NN correlations on predictions of nuclear transparencies for protons, knocked out in high Q2 (e,e'p) reactions

    International Nuclear Information System (INIS)

    Rinat, A.S.; Taragin, M.F.

    1996-01-01

    We study the transparency T of nuclei for nucleons knocked out in high-energy semi-inclusive (e,e'p) reactions, using an improved theoretical input, discussed by Nikolaev et al. We establish that neglect of NN correlations between the knocked-out and core nucleons reduces nuclear transparencies by ∼15 % for light, to ∼10% for heavy nuclei. About the same is predicted for transparencies, integrated over the transverse or longitudinal momentum of the outgoing proton. Hadron dynamics predicts a roughly constant T beyond Q 2 ∼2 GeV 2 , whereas for all targets the largest measured data point Q 2 =6.7 GeV 2 appears to lie above that plateau. Large error bars on those data points preclude a conclusion regarding the onset of colour transparency. (orig.)

  2. Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-Out System

    National Research Council Canada - National Science Library

    Czarneski, Jennifer

    2000-01-01

    .... Mice that carried the Mtv-17 Cre fusion vector would then be mated to mice that had the p53 locus flanked by loxP sites, resulting in the tissue-specific loss of p53 in the mammary gland only. The prediction is that these animals would only develop mammary and not other tumors. We believed that the cre-transgenic mice would also prove useful for other investigators in the mammary gland development and tumorigenesis field.

  3. Energy dependence of the cross section of fast deuteron knock-out from Li, Be, and C by 380 to 665 MeV protons

    Energy Technology Data Exchange (ETDEWEB)

    Komarov, V I; Kosarev, G E; Reshetnikov, G P; Savchenko, O V; Tesh, Z

    1974-12-31

    The high energy parts of the spectra of fast deuterons, which are knocked out from Li, Be and C targets by protons at a 5.5 deg lab. angle with proton energies of 666, 578, 484 and 382 MeV were measured. The cross sections of quasi-elastic deuteron knock-out obtained are compared with the corresponding cross sections of elastic pd-scattering at energies mentioned above. The evaluations of the effective number of two-nucleon clusters are discussed, which have been obtained taking into account (in the Glauber approximation) the incident proton and knocked-out deuteron interactions with nuclear nucleons. The results show the common behavior of the scattering mechanism responsible for elastic pd- and quasi-elastic proton backward scattering with large momentum transfer to two-nucleon clusters. The energy dependence of the deuteron production cross section at the energy kinematically corresponding to the p + N yields d + pi process on tanget nucleons is close to that of the cross section for the p + p yields d + pi /sup +/ process. (auth)

  4. Generación de un modelo knock-out del gen SCN1A en Drosophila melanogaster para el estudio del síndrome de Dravet.

    OpenAIRE

    PLANELLS CÁRCEL, ANDRÉS

    2017-01-01

    [ES] El Síndrome de Dravet (SD) es una enfermedad rara infantil que se manifiesta en crisis epilépticas a temprana edad y provoca un deterioro cognitivo y conductual. Esta enfermedad es causada por mutaciones dominantes en el gen SCN1A. Este trabajo se centra en la generación de un modelo knock-out (KO) del gen paralytic en Drosophila melanogaster, homólogo al gen SCN1A en humanos, para su aplicación en el estudio del SD. A la vez se ha estudiado la conducta de cepas sensibles ...

  5. Characterisation of enterocolitis in the piroxicam-accelerated interleukin-10 knock out mouse--a model mimicking inflammatory bowel disease.

    Science.gov (United States)

    Holgersen, Kristine; Kvist, Peter Helding; Markholst, Helle; Hansen, Axel Kornerup; Holm, Thomas Lindebo

    2014-02-01

    In inflammatory bowel disease a defective mucosal barrier, a dysregulated immune response and an excessive reactivity against the gut microbiota are assumed to cause a breakdown of the intestinal homeostasis and lead to chronic inflammation. Piroxicam treatment is a method for induction of colitis in IL-10 k.o. mice, which integrates a dysfunction of both the intestinal barrier and the immune system. However, the translational value of this model has not been thoroughly clarified. To characterise the piroxicam-accelerated colitis (PAC) IL-10 k.o. model with respect to clinical features, pathogenic mechanisms and its ability to respond to existing therapies. The PAC IL-10k.o. model was established on a C57BL/6J background and the clinical manifestations, immunological mechanisms and efficacy of ampicillin and anti-IL-12/23p40 treatment were assessed. The PAC IL-10 k.o. mice developed weight loss and diarrhoea, and colonoscopy revealed a thickened granulomatous mucosa. Histological evaluation of ileum and colon showed Crohn's disease-like changes with pronounced hyperplasia and focal transmural inflammation. Ileitis was also observed in piroxicam treated wild type mice. The total number of neutrophils, monocytes and natural killer cells was elevated in the blood compared to IL-10 k.o. and wild type mice, indicating a role of the innate immune system in the pathogenesis. These findings were supported by analyses of the intestinal cytokine profile. Ampicillin and anti-IL-12/23p40 treatment significantly suppressed disease in the model. The PAC IL-10 k.o. model resembles several features of Crohn's disease and could be a useful in vivo model in preclinical research. © 2013 Elsevier B.V. All rights reserved.

  6. Platlet Rich Plasma (PRP) Improves Fat Grafting Outcomes.

    Science.gov (United States)

    Modarressi, Ali

    2013-01-01

    Autologous fat transfer offers many qualities of a ideal soft tissue filler. Main advantages of fat grafting ensue from the fact that the lipoaspirate tissue is an abundant source of regenerative pluripotential cells. However, the reported rates of fat cell survival vary greatly in the medical literature (10-90%). Different techniques of harvesting, processing, and reinjecting the fat cells are so claimed to be responsible for these differences, without any agreement concerning the best way to process. To address this important disadvantage, we propose the addition of autologous platelet rich plasma (PRP) which is known as a natural reservoir of growth factors stimulating tissue repair and regeneration. This approach is completely autologous and immediately employed without any type of preconditioning. Platelets rich plasma (PRP) preparation included bleeding of 8 ml of blood from patient's peripheral vein in Regen Lab© tubes containing sodium citrate anticoagulant. The whole blood was centrifugated at 1500 g during 3 min. As Regen-tubes contained a special gel separator, 99 % of red blood cells were discarded from the plasma at the bottom of the gel, and >90% of platelets were harvested in 4 ml of plasma on the top of the gel, called the platelet-rich plasma (PRP). The purified fat prepared by Coleman technique was mixed with different amount of PRP for in vitro, in vivo (mice) and clinical experiments: >50% of PRP for skin rejuvenation, superficial scars correction, infraorbital region, ..., and for 20% of PRP with 80% of purified fat for deep filler indication (nasolabial folds, lips, or soft tissue defect). In vitro studies demonstrated that PRP increased fat cells survival rate and stem cells differentiation. Animal models showed that fat graft survival rate was significantly increased by addition of PRP. Several clinical cases confirmed the improvement of wound healing and fat grafting survival in facial reconstruction and aesthetic cases by association of

  7. Hyperuricemic PRP in Tendon Cells

    Directory of Open Access Journals (Sweden)

    I. Andia

    2014-01-01

    Full Text Available Platelet-rich plasma (PRP is injected within tendons to stimulate healing. Metabolic alterations such as the metabolic syndrome, diabetes, or hyperuricemia could hinder the therapeutic effect of PRP. We hypothesise that tendon cells sense high levels of uric acid and this could modify their response to PRP. Tendon cells were treated with allogeneic PRPs for 96 hours. Hyperuricemic PRP did not hinder the proliferative actions of PRP. The gene expression pattern of inflammatory molecules in response to PRP showed absence of IL-1b and COX1 and modest expression of IL6, IL8, COX2, and TGF-b1. IL8 and IL6 proteins were secreted by tendon cells treated with PRP. The synthesis of IL6 and IL8 proteins induced by PRP is decreased significantly in the presence of hyperuricemia (P = 0.017 and P = 0.012, resp.. Concerning extracellular matrix, PRP-treated tendon cells displayed high type-1 collagen, moderate type-3 collagen, decorin, and hyaluronan synthase-2 expression and modest expression of scleraxis. Hyperuricemia modified the expression pattern of extracellular matrix proteins, upregulating COL1 (P = 0.036 and COMP (P = 0.012 and downregulating HAS2 (P = 0.012. Positive correlations between TGF-b1 and type-1 collagen (R = 0.905, P = 0.002 and aggrecan (R = 0.833, P = 0.010 and negative correlations between TGF-b1 and IL6 synthesis (R = −0.857, P = 0.007 and COX2 (R = −0.810, P = 0.015 were found.

  8. Mouse-hamster chimeric prion protein (PrP) devoid of N-terminal residues 23-88 restores susceptibility to 22L prions, but not to RML prions in PrP-knockout mice.

    Science.gov (United States)

    Uchiyama, Keiji; Miyata, Hironori; Yano, Masashi; Yamaguchi, Yoshitaka; Imamura, Morikazu; Muramatsu, Naomi; Das, Nandita Rani; Chida, Junji; Hara, Hideyuki; Sakaguchi, Suehiro

    2014-01-01

    Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.

  9. Broken or knocked out tooth

    Science.gov (United States)

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

  10. Can PRP effectively treat injured tendons?

    Science.gov (United States)

    Wang, James H-C

    2014-01-01

    PRP is widely used to treat tendon and other tissue injuries in orthopaedics and sports medicine; however, the efficacy of PRP treatment on injured tendons is highly controversial. In this commentary, I reason that there are many PRP- and patient-related factors that influence the outcomes of PRP treatment on injured tendons. Therefore, more basic science studies are needed to understand the mechanism of PRP on injured tendons. Finally, I suggest that better understanding of the PRP action mechanism will lead to better use of PRP for the effective treatment of tendon injuries in clinics.

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

    Directory of Open Access Journals (Sweden)

    Jacqueline Araújo Fiuza

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  13. Search for anti p-nucleus states using the (anti p,p) knock-out reaction at 600 MeV/c

    International Nuclear Information System (INIS)

    Aslanides, E.; Drake, D.M.; Peng, J.C.; Garreta, D.; Birien, P.; Bruge, G.; Catz, H.; Chaumeaux, A.; Janouin, S.; Legrand, D.; Lemaire, M.C.; Mayer, B.; Pain, J.; Perrot, F.

    1987-01-01

    The knock-out reaction A(anti p,p)X has been used to search for narrow anti p-nucleus states. The experiment was performed using the 600 MeV/c antiproton beam at LEAR and the high-resolution and large-acceptance magnetic spectrometer SPES II. The A-dependence of the annihilation-induced proton spectra has been studied on 2 H, 6 Li, 12 C, 63 Cu, 208 Pb and 209 Bi. The quasi-free elastic anti pp scattering observed in the lighter targets, and the comparison with the free anti pp scattering, also observed in this experiment, determine an effective proton number N eff for 1s- and 1p-shell protons. No evidence for narrow bound or resonant anti p-nucleus states could be found. Upper limits for their production are one order of magnitude lower than certain theoretical predictions, but consistent with the properties of the anti p-nucleus interaction, as established from recent elastic and inelastic scattering as well as from studies of antiprotonic atoms. (orig.)

  14. Development of a one-step gene knock-out and knock-in method for metabolic engineering of Aureobasidium pullulans.

    Science.gov (United States)

    Guo, Jian; Wang, Yuanhua; Li, Baozhong; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

    2017-06-10

    Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40bp length and 10μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    OpenAIRE

    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 ...

  16. P55 tumour necrosis factor receptor in bone marrow-derived cells promotes atherosclerosis development in low-density lipoprotein receptor knock-out mice

    NARCIS (Netherlands)

    Xanthoulea, Sofia; Gijbels, Marion J. J.; van der Made, Ingeborg; Mujcic, Hilda; Thelen, Melanie; Vergouwe, Monique N.; Ambagts, Matheus H. C.; Hofker, Marten H.; de Winther, Menno P. J.

    2008-01-01

    Tumour necrosis factor (TNF) is a pivotal pro-inflammatory cytokine with a clear pathogenic role in many chronic inflammatory diseases, and p55 TNF receptor (TNFR) mediates the majority of TNF responses. The aim of the current study was to investigate the role of p55 TNFR expression in bone

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

    DEFF Research Database (Denmark)

    Matheu, Victor; Treschow, Alexandra; Teige, Ingrid

    2005-01-01

    BACKGROUND: CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-beta in the immunomodulatory effects o...

  18. Phase diagram of Pr-P system

    International Nuclear Information System (INIS)

    Mironov, K.E.

    1981-01-01

    An area of the Pr-P system, adjoining to the Pr ordinate, is plotted up by the DTA method. Presence of P solid solution in Pr is established. Data on thermal stability of PrP, PrP 2 , PrP 5 and PrP 7 are generalized. The diagram of phase transformations in Pr-P system is plotted up proceeding from the whole complex of the data, presented. A supposition is made on a possible formation of solid solutions between the highest polyphosphide and phosphorus [ru

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

    Science.gov (United States)

    Sabri, Suriana; Steen, Jennifer A; Bongers, Mareike; Nielsen, Lars K; Vickers, Claudia E

    2013-06-24

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

  20. Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS.

    Science.gov (United States)

    Asante, Emmanuel A; Grimshaw, Andrew; Smidak, Michelle; Jakubcova, Tatiana; Tomlinson, Andrew; Jeelani, Asif; Hamdan, Shyma; Powell, Caroline; Joiner, Susan; Linehan, Jacqueline M; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

    2015-07-01

    Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino aci...... knock-out mutants, where the DLGR-2 gene is interrupted by a P element insertion, die around the time of hatching. This finding, together with the expression data, strongly suggests that DLGR-2 is exclusively involved in development....

  3. Hochaufgelöste Magnetresonanz-Bildgebung der Mäuseaorta zur Bestimmung der Dynamik funktioneller Parameter durch Laufrad-Training bei ApoE-Knock-Out-Mäusen

    OpenAIRE

    Offenberger, Wolfgang

    2009-01-01

    Einführung: Atherosklerose ist eine führende Ursache von Morbidität und Mortalität weltweit. Die ApoE-Knock-Out-Maus (ApoE-/-) ist das wichtigste Tiermodell für das Studium der Atherosklerose und von Interventionen auf diese Erkrankung. Mittels hochaufgelöster Magnet-Resonanz-Bildgebung ist es möglich, eine nicht-invasive in-vivo Gefäß-Charakterisierung bei Mäusen durchzuführen. In dieser Arbeit wurden die Auswirkungen von Sport auf die Gefäßfunktion der Aorta ascendens und abdominalis bei Ap...

  4. PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

    Science.gov (United States)

    Barron, Rona M; King, Declan; Jeffrey, Martin; McGovern, Gillian; Agarwal, Sonya; Gill, Andrew C; Piccardo, Pedro

    2016-10-01

    Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aβ), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre

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

    Science.gov (United States)

    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

  6. Hdac6 knock-out increases tubulin acetylation but does not modify disease progression in the R6/2 mouse model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Anna Bobrowska

    Full Text Available Huntington's disease (HD is a progressive neurodegenerative disorder for which there is no effective disease modifying treatment. Following-on from studies in HD animal models, histone deacetylase (HDAC inhibition has emerged as an attractive therapeutic option. In parallel, several reports have demonstrated a role for histone deacetylase 6 (HDAC6 in the modulation of the toxicity caused by the accumulation of misfolded proteins, including that of expanded polyglutamine in an N-terminal huntingtin fragment. An important role for HDAC6 in kinesin-1 dependent transport of brain-derived neurotrophic factor (BDNF from the cortex to the striatum has also been demonstrated. To elucidate the role that HDAC6 plays in HD progression, we evaluated the effects of the genetic depletion of HDAC6 in the R6/2 mouse model of HD. Loss of HDAC6 resulted in a marked increase in tubulin acetylation throughout the brain. Despite this, there was no effect on the onset and progression of a wide range of behavioural, physiological, molecular and pathological HD-related phenotypes. We observed no change in the aggregate load or in the levels of soluble mutant exon 1 transprotein. HDAC6 genetic depletion did not affect the efficiency of BDNF transport from the cortex to the striatum. Therefore, we conclude that HDAC6 inhibition does not modify disease progression in R6/2 mice and HDAC6 should not be prioritized as a therapeutic target for HD.

  7. PRP and Articular Cartilage: A Clinical Update

    Science.gov (United States)

    Rossi, Roberto; Castoldi, Filippo; Michielon, Gianni

    2015-01-01

    The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory. PMID:26075244

  8. PRP and Articular Cartilage: A Clinical Update

    Directory of Open Access Journals (Sweden)

    Antonio Marmotti

    2015-01-01

    Full Text Available The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory.

  9. Portable Radiation Package (PRP) Instrument Handbook

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, R Michael [Remote Measurements and Research Company, Seattle, WA (United States)

    2017-08-03

    The Portable Radiation Package (PRP) was developed to provide basic radiation information in locations such as ships at sea where proper exposure is remote and difficult, the platform is in motion, and azimuth alignment is not fixed. Development of the PRP began at Brookhaven National Laboratory (BNL) in the mid-1990s and versions of it were deployed on ships in the U.S. Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) Climate Research Facility’s Nauru-99 project. The PRP was deployed on ships in support of the National Aeronautics and Space Administration (NASA) Sensor Intercomparison for Marine Biological and Interdisciplinary Ocean Studies (SIMBIOS) program. Over the years the measurements have remained the same while the post-processing data analysis, especially for the FRSR, has evolved. This document describes the next-generation Portable Radiation Package (PRP2) that was developed for the DOE ARM Facility, under contract no. 9F-31462 from Argonne National Laboratory (ANL). The PRP2 has the same scientific principles that were well validated in prior studies, but has upgraded electronic hardware. The PRP2 approach is completely modular, both in hardware and software. Each sensor input is treated as a separate serial stream into the data collection computer. In this way the operator has complete access to each component of the system for purposes of error checking, calibration, and maintenance. The resulting system is more reliable, easier to install in complex situations, and more amenable to upgrade.

  10. Knocking out IL-6 by vaccination

    DEFF Research Database (Denmark)

    Galle, Pia; Hougs, Lotte; Barington, Torben

    2004-01-01

    Inappropriate expression of IL-6 plays a role in various inflammatory conditions, degenerative diseases, and cancers. Several model systems have been developed that can specifically block IL-6-receptor interactions. Here we present a simple and highly effective approach based on vaccination with ...

  11. Mice lacking Brinp2 or Brinp3, or both, exhibit behaviours consistent with neurodevelopmental disorders

    Directory of Open Access Journals (Sweden)

    Susie Ruth Berkowicz

    2016-10-01

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

  12. PRP: a FORTRAN IV interactive plotting program

    Science.gov (United States)

    Andrew, A. S.; Linde, J.

    A computer program, PRP, has been designed to plot any arithmetic combination selected from a set of major and trace element data on a y- x graph. y and x are defined and entered as a program string (y, x) which is interpreted sequentially. Operators ( +, -, ∗, /, ( unary) , square root, log 10, In c, antilog 10, exponential, integer, absolute value, (,),,) and integer or real numbers may be included. Axis lengths and scales are determined by the user. Five different plotting symbols are available.

  13. Of Men and Mice: Modeling the Fragile X Syndrome

    Science.gov (United States)

    Dahlhaus, Regina

    2018-01-01

    The Fragile X Syndrome (FXS) is one of the most common forms of inherited intellectual disability in all human societies. Caused by the transcriptional silencing of a single gene, the fragile x mental retardation gene FMR1, FXS is characterized by a variety of symptoms, which range from mental disabilities to autism and epilepsy. More than 20 years ago, a first animal model was described, the Fmr1 knock-out mouse. Several other models have been developed since then, including conditional knock-out mice, knock-out rats, a zebrafish and a drosophila model. Using these model systems, various targets for potential pharmaceutical treatments have been identified and many treatments have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients have failed thus far. In this review, I will discuss underlying difficulties and address potential alternatives for our future research. PMID:29599705

  14. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges.

    Science.gov (United States)

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-10-20

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group.

  15. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges

    International Nuclear Information System (INIS)

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-01-01

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group. (paper)

  16. Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

    Science.gov (United States)

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-01-01

    Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

  17. Mice knocked out for the primary brain calcification associated gene Slc20a2 show unimpaired pre-natal survival but retarded growth and nodules in the brain that grow and calcify over time

    DEFF Research Database (Denmark)

    Jensen, Nina; Schrøder, Henrik Daa; Kildall Hejbøl, Eva

    2018-01-01

    Brain calcification of especially the basal ganglia characterises primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; about 40% of patien...

  18. PRP as an Adjunct to Rotator Cuff Tendon Repair.

    Science.gov (United States)

    Barber, F Alan

    2018-06-01

    Arthroscopic rotator cuff repair is a commonly performed repair. Technical developments provide surgeons the tools to create biomechanically robust repairs. How can the biological response mirror the strong and stable surgery? Platelet-rich plasma (PRP) is a supraphysiological platelet concentration which may positively augment rotator cuff healing. Not all PRPs are the same. High leukocyte levels and thrombin activation may be detrimental to tendon healing. Thrombin activation triggers an immediate release of growth factors and may actually inhibit some parts of the healing response. Clear differences exist between liquid PRP (products released within hours after activation) and solid fibrin PRP which slowly releases factors over days. The heterogenicity data and grouping liquid and solid PRP together make systematic reviews confusing. Solid PRP fibrin constructs are often associated with increased tendon healing. PRP fibrin matrix offers the greatest promise for improving clinical success after rotator cuff tendon repair.

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

    Directory of Open Access Journals (Sweden)

    Cheng Qu

    2016-11-01

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

  20. Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

    Science.gov (United States)

    Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

    2016-02-19

    The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. PRP in OA knee - update, current confusions and future options.

    Science.gov (United States)

    Dhillon, Mandeep S; Patel, Sandeep; John, Rakesh

    2017-01-01

    Positive results have been uniformly observed by various researchers for platelet-rich plasma (PRP) in early osteoarthritis (OA) knee in the past few years. PRP has clearly demonstrated its supremacy in comparison to hyaluronic acid (HA) and placebo in various clinical trials and is undoubtedly the best option available for symptomatic treatment in early OA. The release of growth factors from PRP occurs immediately and lasts for around three weeks and the clinical effect tends to wane down by the end of the year. Prolonged and sustained release of growth factors from platelets could possibly help in much better biological healing and sustained clinical effects. PRP in combination with biocompatible carriers could be one way of achieving this. Gelatin hydrogel PRP and chitosan PRP seem to be promising based on early in vitro studies and animal studies. PRP in combination with hyaluronic acid also seems to be additive. This article intends to discuss the present status of the PRP, confusions surrounding its use, upcoming trends and ideas for improvising PRP for use early OA knees based on available evidence. © The Authors, published by EDP Sciences, 2017.

  2. Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

    Science.gov (United States)

    Khan, M. Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R.; Pai, Emil F.; Chakrabartty, Avijit

    2010-01-01

    Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrPSc). Exposure to PrPSc from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer–octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease. PMID:21041683

  3. Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

    Science.gov (United States)

    Khan, M Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R; Pai, Emil F; Chakrabartty, Avijit

    2010-11-16

    Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

  4. PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

    Science.gov (United States)

    Juma, Almas R; Grommen, Sylvia V H; O'Bryan, Moira K; O'Connor, Anne E; Merriner, D Jo; Hall, Nathan E; Doyle, Stephen R; Damdimopoulou, Pauliina E; Barriga, Daniel; Hart, Adam H; Van de Ven, Wim J M; De Groef, Bert

    2017-07-13

    Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

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

    Directory of Open Access Journals (Sweden)

    Ilgiz A Mufazalov

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

  6. Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

    Science.gov (United States)

    Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

    2017-05-01

    Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  7. Na+/K+-ATPase is present in scrapie-associated fibrils, modulates PrP misfolding in vitro and links PrP function and dysfunction.

    Directory of Open Access Journals (Sweden)

    James F Graham

    Full Text Available Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C, to the disease-associated form, PrP(Sc, through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C and PrP(Sc isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C or to provide a scaffold ensuring correct alignment of PrP(C and PrP(Sc during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+/K(+-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+/K(+-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C and Na(+/K(+-ATPase have previously been reported in astrocytes, our data highlight this molecule as

  8. PrPST, a Soluble, Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

    Science.gov (United States)

    Frid, Kati; Binyamin, Orli; Gabizon, Ruth

    2013-01-01

    While the conversion of PrPC into PrPSc in the transmissible form of prion disease requires a preexisting PrPSc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrPST), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrPST as in TgMHu2ME199K mice, and “classical” PrPSc as in infectious prion diseases, coincide in the patient's post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition. PMID:23922744

  9. PrP(ST), a soluble, protease resistant and truncated PrP form features in the pathogenesis of a genetic prion disease.

    Science.gov (United States)

    Friedman-Levi, Yael; Mizrahi, Michal; Frid, Kati; Binyamin, Orli; Gabizon, Ruth

    2013-01-01

    While the conversion of PrP(C) into PrP(Sc) in the transmissible form of prion disease requires a preexisting PrP(Sc) seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP(ST)), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP(ST) as in TgMHu2ME199K mice, and "classical" PrP(Sc) as in infectious prion diseases, coincide in the patient's post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.

  10. PrP(ST, a soluble, protease resistant and truncated PrP form features in the pathogenesis of a genetic prion disease.

    Directory of Open Access Journals (Sweden)

    Yael Friedman-Levi

    Full Text Available While the conversion of PrP(C into PrP(Sc in the transmissible form of prion disease requires a preexisting PrP(Sc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP(ST, a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP(ST as in TgMHu2ME199K mice, and "classical" PrP(Sc as in infectious prion diseases, coincide in the patient's post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.

  11. [Platelet rich plasma (PRP): potentialities and techniques of extraction].

    Science.gov (United States)

    Pacifici, L; Casella, F; Maggiore, C

    2002-01-01

    This paper describes the various techniques of platelet-rich plasma (PRP) extraction codified in recent years and their use potential is evaluated. PRP is one of the techniques with which at the moment it is attempted to modulate and facilitate the cure of a wound. The use of PRP is based on the theoretical premise that by concentrating platelets the effects of the growth factors (PDGF, TGF-beta, IGF-I and -II) so released will be increased. Marx's original technique is described above all. This prescribes the sampling of a unit of blood (450-500 ml) and the use of a cell separator. We then analysed the technique of Marx and Hannon in which the quantity of blood sampled is reduced to 150 ml, and the two simplified techniques of the Sacchi and Bellanda group. Finally, a new PRP extraction technique is described. We conclude that platelet gel allows access to autologous growth factors which by definition are neither toxic nor immunogenic and are capable of accelerating the normal processes of bone regeneration. PRP can thus be considered a useful instrument for increasing the quality and final quantity of regenerated bone in oral and maxillo-facial surgery operations.

  12. Long-term trends in invasive Haemophilus influenzae type B disease among indigenous Australian children following use of PRP-OMP and PRP-T vaccines.

    Science.gov (United States)

    Menzies, Robert Ian; Bremner, Kyla Margaret; Wang, Han; Beard, Frank Hudson; McIntyre, Peter Bruce

    2015-06-01

    Among indigenous populations with high incidence and early onset of invasive Haemophilus influenzae type b (Hib) disease, PRP-OMP vaccines are used in the United States and PRP-T vaccines in Canada. In Australia, PRP-OMP vaccines were exclusively used in indigenous children from 1993 until they were replaced by PRP-T between late 2005 and 2009. Analytic descriptive study of 20 years of enhanced surveillance data (1993-2013) for invasive Hib disease in Australian children PRP-OMP period (1993-1996) to 6.2 (95% CI: 4.0, 9.2) and 4.7 (95% CI: 1.7, 10.3) in the later PRP-OMP (1996-2009) and PRP-T periods (2009-2013), respectively. The indigenous:nonindigenous incidence rate ratio increased to 43 (95% CI: 16, 145) and 58 (95% CI: 7, 2660) in the later PRP-OMP and PRP-T periods, respectively, more than 10-fold higher than in lesser-incidence Australian regions. We found no change in Hib incidence among indigenous Australian children living in high-incidence regions in the first 4 years following a change to PRP-T-containing combination vaccines. This may be of relevance to North American indigenous populations characterized by suboptimal living conditions and young age of onset for whom PRP-OMP continues to be recommended, such as Alaska Natives.

  13. Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

    OpenAIRE

    Alkan , Manal; Machavoine , François; Rignault , Rachel; Dam , Julie; Dy , Michel; Thieblemont , Nathalie

    2015-01-01

    International audience; Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC −/− m...

  14. Astrocytic β2 Adrenergic Receptor Gene Deletion Affects Memory in Aged Mice.

    Directory of Open Access Journals (Sweden)

    Cathy Joanna Jensen

    Full Text Available In vitro and in vivo studies suggest that the astrocytic adrenergic signalling enhances glycogenolysis which provides energy to be transported to nearby cells and in the form of lactate. This energy source is important for motor and cognitive functioning. While it is suspected that the β2-adrenergic receptor on astrocytes might contribute to this energy balance, it has not yet been shown conclusively in vivo. Inducible astrocyte specific β2-adrenergic receptor knock-out mice were generated by crossing homozygous β2-adrenergic receptor floxed mice (Adrb2flox and mice with heterozygous tamoxifen-inducible Cre recombinase-expression driven by the astrocyte specific L-glutamate/L-aspartate transporter promoter (GLAST-CreERT2. Assessments using the modified SHIRPA (SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment test battery, swimming ability test, and accelerating rotarod test, performed at 1, 2 and 4 weeks, 6 and 12 months after tamoxifen (or vehicle administration did not reveal any differences in physical health or motor functions between the knock-out mice and controls. However deficits were found in the cognitive ability of aged, but not young adult mice, reflected in impaired learning in the Morris Water Maze. Similarly, long-term potentiation (LTP was impaired in hippocampal brain slices of aged knock-out mice maintained in low glucose media. Using microdialysis in cerebellar white matter we found no significant differences in extracellular lactate or glucose between the young adult knock-out mice and controls, although trends were detected. Our results suggest that β2-adrenergic receptor expression on astrocytes in mice may be important for maintaining cognitive health at advanced age, but is dispensable for motor function.

  15. Intra-articular laser treatment plus Platelet Rich Plasma (PRP) significantly reduces pain in many patients who had failed prior PRP treatment

    Science.gov (United States)

    Prodromos, Chadwick C.; Finkle, Susan; Dawes, Alexander; Dizon, Angelo

    2018-02-01

    INTRODUCTION: In our practice Platelet Rich Plasma (PRP) injections effectively reduce pain in most but not all arthritic patients. However, for patients who fail PRP treatment, no good alternative currently exists except total joint replacement surgery. Low level laser therapy (LLLT) on the surface of the skin has not been helpful for arthritis patients in our experience. However, we hypothesized that intra-articular laser treatment would be an effective augmentation to PRP injection and would increase its efficacy in patients who had failed prior PRP injection alone. METHODS: We offered Intra-articular Low Level Laser Therapy (IAL) treatment in conjunction with repeat PRP injection to patients who had received no benefit from PRP injection alone at our center. They were the treatment group. They were not charged for PRP or IAL. They also served as a historical control group since they had all had failed PRP treatment alone. 28 patients (30 joints) accepted treatment after informed consent. 22 knees, 4 hips, 2 shoulder glenohumeral joints and 1 first carpo-metacarpal (1st CMC) joint were treated RESULTS: All patients were followed up at 1 month and no adverse events were seen from the treatment. At 6 months post treatment 46% of patients had good outcomes, and at 1 year 17% still showed improvement after treatment. 11 patients failed treatment and went on to joint replacement. DISCUSSION: A single treatment of IAL with PRP salvaged 46% of patients who had failed PRP treatment alone, allowing avoidance of surgery and good pain control.

  16. Purification and Fibrillation of Full-Length Recombinant PrP

    OpenAIRE

    Makarava, Natallia; Baskakov, Ilia V.

    2012-01-01

    Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt–Jacob disease, fatal familial insomnia, and Gerstmann–Straussler–Sheinker disease. Certain applications in prion area require recombinant PrP of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian PrP. This protocol has been proved to yield PrP of extremely high purity that lac...

  17. PRP: The Proven Solution for Cleaning Up Oil Spills

    Science.gov (United States)

    2006-01-01

    The basic technology behind PRP is thousands of microcapsules, tiny balls of beeswax with hollow centers. Water cannot penetrate the microcapsule s cell, but oil is absorbed right into the beeswax spheres as they float on the water s surface. This way, the contaminants, chemical compounds that originally come from crude oil such as fuels, motor oils, or petroleum hydrocarbons, are caught before they settle. PRP works well as a loose powder for cleaning up contaminants in lakes and other ecologically fragile areas. The powder can be spread over a contaminated body of water or soil, and it will absorb contaminants, contain them in isolation, and dispose of them safely. In water, it is important that PRP floats and keeps the oil on the surface, because, even if oil exposure is not immediately lethal, it can cause long-term harm if allowed to settle. Bottom-dwelling fish exposed to compounds released after oil spills may develop liver disease, in addition to reproductive and growth problems. This use of PRP is especially effective for environmental cleanup in sensitive areas like coral reefs and mangroves.

  18. PrP Conformational Transitions Alter Species Preference of a PrP-specific antibody

    NARCIS (Netherlands)

    Zou, W.Q.; Langeveld, J.P.M.; Xiao, X.; Chen, S.; McGeer, P.L.; Yuan, J.; Payne, M.C.; Kang, H.E.; McGeehan, J.M.; Sy, M.S.; Greenspan, N.S.; Kaplan, D.; Wang, G.X.; Parchi, P.; Hoover, E.A.; Kneale, G.; Telling, G.; Surewicz, W.; Kong, Q.; Guo, J.

    2010-01-01

    The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109–112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from

  19. Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep.

    Science.gov (United States)

    Silva, Christopher J; Erickson-Beltran, Melissa L; Hui, Colleen; Badiola, Juan José; Nicholson, Eric M; Requena, Jesús R; Bolea, Rosa

    2017-01-03

    Scrapie is a prion (PrP Sc ) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep's normal cellular prion protein (PrP C ). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M 132 LGSXMSRPL 141 (X = A or V), Y 153 XENMY 158 (X,= H or R), and Y 166 RPVDXY 172 (X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrP C or PrP Sc . These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrP Sc isolated from sheep heterozygous for their PrP C proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrP Sc . This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrP Sc and samples containing only the PK-resistant PrP Sc . These results show that heterozygous animals contain PrP Sc that is composed of significant amounts of both PrP polymorphisms.

  20. Identification of PrP sequences essential for the interaction between the PrP polymers and Aβ peptide in a yeast-based assay

    OpenAIRE

    Rubel, Aleksandr A; Ryzhova, Tatyana A; Antonets, Kirill S; Chernoff, Yury O; Galkin, Alexey P

    2013-01-01

    Alzheimer disease is associated with the accumulation of oligomeric amyloid β peptide (Aβ), accompanied by synaptic dysfunction and neuronal death. Polymeric form of prion protein (PrP), PrPSc, is implicated in transmissible spongiform encephalopathies (TSEs). Recently, it was shown that the monomeric cellular form of PrP (PrPC), located on the neuron surface, binds Aβ oligomers (and possibly other β-rich conformers) via the PrP23–27 and PrP90–110 segments, acting as Aβ receptor. On the other...

  1. Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

    OpenAIRE

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M.; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligas...

  2. The knocked-out erythrocyte sedimentation rate: periodontal abscess.

    Science.gov (United States)

    Sevinc, Alper; Bayindir, Yasar; But, Ayse

    2008-01-01

    The erythrocyte sedimentation rate (ESR) is a common but nonspecific test that is often used as an indicator of active disease. Infection of dental origin may be responsible for a number of cases in unresolved elevated ESR and fever etiology. Dental sepsis is the one of the potential causes of persistent fever that can escape detection. An 18-year-old female patient was admitted to the emergency room with complaints of headache, fever, nausea, and vomiting for the past four days. Erythrocyte sedimentation rate was 110 mm/h. She was started empirically on antibiotic treatment as no etiology was found. Four days later, while searching for the etiology of the fever, the patient experienced an acute pain in association with localizing symptoms in two decayed teeth. Oral examination revealed abscess formation in both teeth. Teeth were extracted and ESR was decreased to 95 mm/h on the day of the second extraction and to 60, 35, and 10 mm/h taken weekly. During the follow-up, she was in good health with no fever seen 3 months after treatment and her ESR was 15 mm/h. Dental infection should be considered as an unusual but very treatable cause of pyrexia of unknown origin.

  3. Knocking out knotweed: research pins down a rogue invasive

    Science.gov (United States)

    Natasha Vizcarra; Shannon Claeson

    2015-01-01

    Bohemian knotweed spreads aggressively along rivers. This invasive weed chokes waterways, displaces native plants, erodes riverbanks, and keeps tree seedlings from growing. Communities in the Pacific Northwest spend millions of dollars to eradicate it on the assumption that it harms fish habitats.But knotweed is difficult to kill. It takes...

  4. Distortion effects in pion-induced knock-out reactions

    International Nuclear Information System (INIS)

    Jain, B.K.

    The cross-section for (π + ,π + p) reaction on 12 C is calculated in DWIA at 100 and 180 MeV incident energy. The effect of pion distortion is found to be strong. Around 180 MeV the effect is strongly absorptive while around 10O MeV it is mainly dispersive. (auth.)

  5. Help NCI at Frederick “Knock Out Hunger” | Poster

    Science.gov (United States)

    NCI at Frederick is once again participating in the Feds Feed Families initiative, an annual food drive that addresses severe shortages of non-perishable items in food banks across D.C., Maryland, and Virginia during the summer months, when giving is at its lowest.

  6. Prion Protein Devoid of the Octapeptide Repeat Region Delays Bovine Spongiform Encephalopathy Pathogenesis in Mice.

    Science.gov (United States)

    Hara, Hideyuki; Miyata, Hironori; Das, Nandita Rani; Chida, Junji; Yoshimochi, Tatenobu; Uchiyama, Keiji; Watanabe, Hitomi; Kondoh, Gen; Yokoyama, Takashi; Sakaguchi, Suehiro

    2018-01-01

    Conformational conversion of the cellular isoform of prion protein, PrP C , into the abnormally folded, amyloidogenic isoform, PrP Sc , is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrP C into PrP Sc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/ Prnp 0 / 0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrP Sc ΔOR in their brains. We show here that Tg(PrPΔOR)/ Prnp 0 / 0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrP Sc ΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrP C into PrP Sc after infection with BSE prions. However, Tg(PrPΔOR)/ Prnp 0 / 0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrP Sc ΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/ Prnp 0 / 0 mice than PrP Sc in control wild-type mice. Taken together, these results indicate that the OR region of PrP C could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions. IMPORTANCE Structure-function relationship studies of PrP C conformational conversion into PrP Sc are worthwhile to understand the mechanism of the conversion of PrP C into PrP Sc We show here that, by inoculating Tg(PrPΔOR)/ Prnp 0 / 0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of

  7. Two New PRP Conjugate Gradient Algorithms for Minimization Optimization Models.

    Directory of Open Access Journals (Sweden)

    Gonglin Yuan

    Full Text Available Two new PRP conjugate Algorithms are proposed in this paper based on two modified PRP conjugate gradient methods: the first algorithm is proposed for solving unconstrained optimization problems, and the second algorithm is proposed for solving nonlinear equations. The first method contains two aspects of information: function value and gradient value. The two methods both possess some good properties, as follows: 1 βk ≥ 0 2 the search direction has the trust region property without the use of any line search method 3 the search direction has sufficient descent property without the use of any line search method. Under some suitable conditions, we establish the global convergence of the two algorithms. We conduct numerical experiments to evaluate our algorithms. The numerical results indicate that the first algorithm is effective and competitive for solving unconstrained optimization problems and that the second algorithm is effective for solving large-scale nonlinear equations.

  8. Two New PRP Conjugate Gradient Algorithms for Minimization Optimization Models.

    Science.gov (United States)

    Yuan, Gonglin; Duan, Xiabin; Liu, Wenjie; Wang, Xiaoliang; Cui, Zengru; Sheng, Zhou

    2015-01-01

    Two new PRP conjugate Algorithms are proposed in this paper based on two modified PRP conjugate gradient methods: the first algorithm is proposed for solving unconstrained optimization problems, and the second algorithm is proposed for solving nonlinear equations. The first method contains two aspects of information: function value and gradient value. The two methods both possess some good properties, as follows: 1) βk ≥ 0 2) the search direction has the trust region property without the use of any line search method 3) the search direction has sufficient descent property without the use of any line search method. Under some suitable conditions, we establish the global convergence of the two algorithms. We conduct numerical experiments to evaluate our algorithms. The numerical results indicate that the first algorithm is effective and competitive for solving unconstrained optimization problems and that the second algorithm is effective for solving large-scale nonlinear equations.

  9. Study of PrP - I heterogeneous equilibrium

    International Nuclear Information System (INIS)

    Vasil'eva, I.G.; Mironov, K.E.; Tarasenko, A.D.

    1976-01-01

    Using static methods the authors have measured the equilibrium vapor pressure in the system PrP+I 2 at different temperatures and different initial iodine concentrations. The equilibrium reactions in the system have been determined. The reaction of PrP with iodine is irreversible. The content of PrI 3 and I 2 in the gas phase is negligible. The pressure in the system is determined by the partial pressure of phosphorus

  10. Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-06-01

    Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by

  11. Impact of local anaesthetics and needle calibres used for painless PRP injections on platelet functionality.

    Science.gov (United States)

    Bausset, Olivier; Magalon, Jeremy; Giraudo, Laurent; Louis, Marie-Laure; Serratrice, Nicolas; Frere, Corrine; Magalon, Guy; Dignat-George, Françoise; Sabatier, Florence

    2014-01-01

    The platelet-rich plasma (PRP) is an autologous biotherapy commonly used for its healing properties. Once activated, platelets released a real "cocktail" of growth factor and cytokines implied in numerous regenerative processes. However the impact of medical practices associated to PRP therapeutic use on platelets functionality remains poorly known. we evaluated the in vitro effects of two commonly used local anesthetics (Xylocaine(*) and Naropin(*)) on PRP functionality. We also investigated the quantity and quality of PRP that passed through the smallest gauge needle commercialized. PRP from 9 healthy volunteers were prepared using our previously described home made purification protocol. Platelet aggregation capacity was evaluated by aggregometry assays and the growth factor release was determined by ELISA after platelet activation. We also evaluated the platelet activation status, reactivity and stability of platelets by flow cytometry using the P-selectin expression marker. the association of local anaesthetics with PRP injections resulted in a significant decrease of platelets functionality, assessed by their capacity of aggregating. Local anaesthetics did not interfere with the growth factor release. The different needle sizes and calibres tested for PRP injections did not influence the platelet functionality. the use of local anaesthetics to prevent pain during PRP injections could compromise the therapeutic potential of PRP. These results suggest using carefully local anaesthetics or limiting their use as often is possible. To minimize injection pain, we recommend using 30 G needles. These data will lead to clinical recommendations for painless and controlled PRP injections.

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

    OpenAIRE

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

    2016-01-01

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

  13. Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106-126.

    Science.gov (United States)

    Han, Jun; Song, Qin-Qin; Sun, Peng; Zhang, Jin; Wang, Xu; Song, Juan; Li, Gong-Qi; Liu, Ying-Hui; Mei, Guo-Yong; Shi, Qi; Tian, Chan; Chen, Cao; Gao, Chen; Zhao, Bo; Dong, Xiao-Ping

    2014-02-01

    Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106-126 within PrP and aa 1-38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106-126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106-126 fibrils in vitro. Moreover, the PrP-14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. HGF Mediates the Anti-inflammatory Effects of PRP on Injured Tendons

    Science.gov (United States)

    Zhang, Jianying; Middleton, Kellie K.; Fu, Freddie H.; Im, Hee-Jeong; Wang, James H-C.

    2013-01-01

    Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2. PMID:23840657

  15. HGF mediates the anti-inflammatory effects of PRP on injured tendons.

    Directory of Open Access Journals (Sweden)

    Jianying Zhang

    Full Text Available Platelet-rich plasma (PRP containing hepatocyte growth factor (HGF and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2.

  16. Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

    Science.gov (United States)

    Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu

    2015-04-13

    The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

  17. Cytosolic PrP Can Participate in Prion-Mediated Toxicity

    Science.gov (United States)

    Thackray, Alana M.; Zhang, Chang; Arndt, Tina

    2014-01-01

    ABSTRACT Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCE During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and

  18. Glycan-deficient PrP stimulates VEGFR2 signaling via glycosaminoglycan.

    Science.gov (United States)

    Gao, Zhenxing; Zhang, Huixia; Hu, Fei; Yang, Liheng; Yang, Xiaowen; Zhu, Ying; Sy, Man-Sun; Li, Chaoyang

    2016-06-01

    Whether the two N-linked glycans are important in prion, PrP, biology is unresolved. In Chinese hamster ovary (CHO) cells, the two glycans are clearly not important in the cell surface expression of transfected human PrP. Compared to fully-glycosylated PrP, glycan-deficient PrP preferentially partitions to lipid raft. In CHO cells glycan-deficient PrP also interacts with glycosaminoglycan (GAG) and vascular endothelial growth factor receptor 2 (VEGFR2), resulting in VEGFR2 activation and enhanced Akt phosphorylation. Accordingly, CHO cells expressing glycan-deficient PrP lacking the GAG binding motif or cells treated with heparinase to remove GAG show diminished Akt signaling. Being in lipid raft is critical, chimeric glycan-deficient PrP with CD4 transmembrane and cytoplasmic domains is absent in lipid raft and does not activate Akt signaling. CHO cells bearing glycan-deficient PrP also exhibit enhanced cellular adhesion and migration. Based on these findings, we propose a model in which glycan-deficient PrP, GAG, and VEGFR2 interact, activating VEGFR2 and resulting in changes in cellular behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Purification and Fibrillation of Full-Length Recombinant PrP.

    Science.gov (United States)

    Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V

    2017-01-01

    Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrP Sc and methods for production of PrP Sc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrP Sc for development of prion diagnostics including generation of PrP Sc -specific antibody.

  20. Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress.

    Science.gov (United States)

    Jiang, Qian-Tao; Liu, Tao; Ma, Jian; Wei, Yu-Ming; Lu, Zhen-Xiang; Lan, Xiu-Jin; Dai, Shou-Fen; Zheng, You-Liang

    2011-10-01

    The pre-mRNA processing (Prp1) gene encodes a spliceosomal protein. It was firstly identified in fission yeast and plays a regular role during spliceosome activation and cell cycle. Plant Prp1 genes have only been identified from rice, Sorghum and Arabidopsis thaliana. In this study, we reported the identification and isolation of a novel Prp1 gene from barley, and further explored its expressional pattern by using real-time quantitative RTPCR, promoter prediction and analysis of microarray data. The putative barley Prp1 protein has a similar primary structure features to those of other known Prp1 protein in this family. The results of amino acid comparison indicated that Prp1 protein of barley and other plant species has a highly conserved 30 termnal region while their 50 sequences greatly varied. The results of expressional analysis revealed that the expression level of barley Prp1 gene is always stable in different vegetative tissues, except it is up-regulated at the mid- and late stages of seed development or under the condition of cold stress. This kind of expressional pattern for barley Prp1 is also supported by our results of comparison of microarray data from barley, rice and Arabidopsis. For the molecular mechanism of its expressional pattern, we conclude that the expression of Prp1 gene may be up-regulated by the increase of pre-mRNAs and not be constitutive or ubiquitous.

  1. An introduction to application of Platelet Rich Plasma (PRP in skin rejuvenation

    Directory of Open Access Journals (Sweden)

    Mahnaz Banihashemi

    2014-04-01

    Full Text Available Platelet-rich plasma (PRP is an autologous concentration of human platelets contained in a small volume of plasma characterized by haemostatic and tissue repairing effects. Tissue repairing effects and being enriched by various kind of growth factors, has made them the focus of attention for different procedures. PRP has been effective in bony defects, wound healing and recently for aesthetic procedures in plastic surgery. The purpose of this review is to evaluate and summarize the applications of PRP in the dermatology literature, with particular focus on rejuvenizaton process, advances and limitations of current PRP therapies. We studied literature related to PRP therapy, these include regeneration of soft tissue, skin aging mechanisms, as well as wound healing. Some studies have shown promising results, with favorable outcomes about PRP clinical application for skin rejuvenization. This article summarizes our current understanding regarding photoaging process and the role of PRP in the skin rejuvenization process. PRP has been shown to be useful in skin rejuvenization. Further studies are needed to elucidate both basic and clinical aspects of PRP therapies. In particular, platelet preparation methods, different application methods, platelet mechanism of action in rejuvenation field, interactions with the skin components, long-term efficacy and safety are necessary to be determined.

  2. Performance related pay (PRP) to social workers in Danish Job Centres

    DEFF Research Database (Denmark)

    Flensborg Jensen, Maya; Rosdahl, Anders

    This paper discusses two issues: - Why has some Danish local employment administrations introduced performance related pay (PRP) for social workers while others have not? - Does PRP to social workers imply better efforts to bring long-term recipients of social assistance into employment?......This paper discusses two issues: - Why has some Danish local employment administrations introduced performance related pay (PRP) for social workers while others have not? - Does PRP to social workers imply better efforts to bring long-term recipients of social assistance into employment?...

  3. Prion propagation in cells expressing PrP glycosylation mutants.

    Science.gov (United States)

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  4. Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

    International Nuclear Information System (INIS)

    Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi

    2007-01-01

    Splenocytes of wild-type (Prnp +/+ ) and prion protein gene-deficient (Prnp -/- ) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP C ) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP C and PrPLP/Doppel

  5. Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

    NARCIS (Netherlands)

    Meade-White, K.; Race, B.; Trifilo, M.; Bossers, A.; Favara, C.; Lacasse, R.; Miller, M.; Williams, E.; Oldstone, M.; Race, R.; Chesebro, B.

    2007-01-01

    Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer

  6. Enhanced Osteogenic Differentiation of Mesenchymal Stem Cells on Electrospun PES/PVA/PRP Nanofibrous Scaffolds.

    Science.gov (United States)

    Kashef-Saberi, Mahshid Sadat; Roodbari, Nasim Hayati; Parivar, Kazem; Vakilian, Saeid; Hanee-Ahvaz, Hana

    2018-03-28

    Over the last few decades, great advancements have been achieved in the field of bone tissue engineering (BTE). Containing a great number of growth factors needed in the process of osteogenesis, platelet rich plasma (PRP) has gained a great deal of attention. However, due to the contradictory results achieved in different studies, its effectiveness remains a mystery. Therefore, in this study, we investigated in vitro performance of co-electrospun PRP/poly ether sulfone/poly(vinyl) alcohol (PRP/PES/PVA) composite scaffolds for the osteogenic differentiation of human adipose-derived mesenchymal stem cells. The activated PRP was mixed with PVA solution to be used alongside PES solution for the electrospinning process. Fourier transform infrared spectroscopy, scanning electron microscopy and tensile tests were performed to evaluate the scaffolds. After confirmation of sustained release of protein, osteogenic potential of the co-electrospun PRP/polymer scaffolds was evaluated by measuring relative gene expression, calcium content, and alkaline phosphatase (ALP) activity. Alizarin red and Hematoxylin and Eosin staining were performed as well. The results of ALP activity and calcium content demonstrated the effectiveness of PRP when combined with PRP-incorporated scaffold in comparison with the other tested groups. In addition, the results of tensile mechanical testing indicated that addition of PRP improves the mechanical properties. Taking these results into account, it appears PES/PVA/PRP scaffold treated with PRP 5% enhances osteogenic differentiation most. In conclusion, incorporation of PRP into electrospun PES/PVA scaffold in this study had a positive influence on osteogenic differentiation of AdMSCs, and thus it may have great potential for BTE applications.

  7. Redox induces diverse effects on recombinant human wild-type PrP and mutated PrP with inserted or deleted octarepeats.

    Science.gov (United States)

    Shi, Qi; Chen, Cao; Zhang, Bao-Yun; Zhou, Wei; Xiao, Kang; Dong, Xiao-Ping

    2018-04-01

    Normal prion protein (PrP) contains two cysteines at amino acids 179 and 214, which may form intra‑ and interpeptide disulfide bonds. To determine the possible effects of this disulfide bridge on the biochemical features of PrP, prokaryotic recombinant human wild‑type PrP (PG5), and mutated PrPs with seven extra octarepeats (PG12) or with all five octarepeats removed (PG0), were subjected to redox in vitro. Sedimentation assays revealed a large portion of aggregation in redox‑treated PG5, but not in PG0 and PG12. Circular dichroism analysis detected increased β‑sheet and decreased α‑helix in PG5 subjected to redox, increased random‑coil and decreased β‑sheet in PG0, and increased random‑coil, but limited changes to β‑sheet content, in PG12. Thioflavin T fluorescence tests indicated that fluorescent value was increased in PG5 subjected to redox. In addition, proteinase K (PK) digestions indicated that PK resistance was stronger in PG12 and PG0 compared with in PG5; redox enhanced the PK resistance of all three PrP constructs, particularly PG0 and PG12. These data indicated that formation of a disulfide bond induces marked alterations in the secondary structure and biochemical characteristics of PrP. In addition, the octarepeat region within the PrP peptide markedly influences the effects of redox on the biochemical phenotypes of PrP, thus highlighting the importance of the number of octarepeats in the biological functions of PrP.

  8. Recommendations for the Involvement of Patient Research Partners (PRP) in OMERACT Working Groups. A Report from the OMERACT 2014 Working Group on PRP.

    Science.gov (United States)

    Cheung, Peter P; de Wit, Maarten; Bingham, Clifton O; Kirwan, John R; Leong, Amye; March, Lyn M; Montie, Pam; Scholte-Voshaar, Marieke; Gossec, Laure

    2016-01-01

    Patient participation in research is increasing; however, practical guidelines to enhance this participation are lacking. Specifically within the Outcome Measures in Rheumatology (OMERACT) organization, although patients have participated in OMERACT meetings since 2002, consensus about the procedures for involving patients in working groups has not been formalized. The objective is to develop a set of recommendations regarding patient research partner (PRP) involvement in research working groups. We conducted a systematic literature review on recommendations/guidelines of PRP involvement in research; elaborated a structured consensus process involving multiple participants to develop a set of recommendations; and sought endorsement of recommendations by OMERACT. In the 18 articles included in the literature review, there was general agreement on the broad concepts for recommendations covering PRP involvement in research although they were heterogeneous in detail. Most considered PRP involvement in all phases of research with early engagement, training, and support important, but details on the content were scarce. This review informed a larger consensus-building process regarding PRP inclusion in OMERACT research. Three overarching principles and 8 recommendations were developed, discussed, and refined at OMERACT 2014. The guiding principles were endorsed during the OMERACT plenary session. These recommendations for PRP involvement in OMERACT research reinforce the importance of patient participation throughout the research process as integral members. Although the applicability of the recommendations in other research contexts should be assessed, the generalizability is expected to be high. Future research should evaluate their implementation and their effect on outcome development.

  9. Expressions of pathologic markers in PRP based chondrogenic differentiation of human adipose derived stem cells.

    Science.gov (United States)

    Pakfar, Arezou; Irani, Shiva; Hanaee-Ahvaz, Hana

    2017-02-01

    Optimization of the differentiation medium through using autologous factors such as PRP is of great consideration, but due to the complex, variable and undefined composition of PRP on one hand and lack of control over the absolute regulatory mechanisms in in vitro conditions or disrupted and different mechanisms in diseased tissue microenvironments in in vivo conditions on the other hand, it is complicated and rather unpredictable to get the desired effects of PRP making it inevitable to monitor the possible pathologic or undesired differentiation pathways and therapeutic effects of PRP. Therefore, in this study the probable potential of PRP on inducing calcification, inflammation and angiogenesis in chondrogenically-differentiated cells was investigated. The expressions of chondrogenic, inflammatory, osteogenic and angiogenic markers from TGFβ or PRP-treated cells during chondrogenic differentiation of human adipose-derived stem cells (ADSCs) was evaluated. Expressions of Collagen II (Col II), Aggrecan, Sox9 and Runx2 were quantified using q-RT PCR. Expression of Col II and X was investigated by immunocytochemistry as well. Glycosaminoglycans (GAGs) production was also determined by GAG assay. Possible angiogenic/inflammatory potential was determined by quantitatively measuring the secreted VEGF, TNFα and phosphorylated VEGFR2 via ELISA. In addition, the calcification of the construct was monitored by measuring ALP activity and calcium deposition. Our data showed that PRP positively induced chondrogenesis; meanwhile the secretion of angiogenic and inflammatory markers was decreased. VEGFR2 phosphorylation and ALP activity had a decreasing trend, but tissue mineralization was enhanced upon treating with PRP. Although reduction in inflammatory/angiogenic potential of the chondrogenically differentiated constructs highlights the superior effectiveness of PRP in comparison to TGFβ for chondrogenic differentiation, yet further improvement of the PRP

  10. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    Energy Technology Data Exchange (ETDEWEB)

    Möhlmann, Sina [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Mathew, Rebecca [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Neumann, Piotr; Schmitt, Andreas [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Lührmann, Reinhard [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@uni-goettingen.de [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  11. The composite of bone marrow concentrate and PRP as an alternative to autologous bone grafting.

    Directory of Open Access Journals (Sweden)

    Mohssen Hakimi

    Full Text Available One possible alternative to the application of autologous bone grafts represents the use of autologous bone marrow concentrate (BMC. The purpose of our study was to evaluate the potency of autologous platelet-rich plasma (PRP in combination with BMC. In 32 mini-pigs a metaphyseal critical-size defect was surgically created at the proximal tibia. The animals were allocated to four treatment groups of eight animals each (1. BMC+CPG group, 2. BMC+CPG+PRP group, 3. autograft group, 4. CPG group. In the BMC+CPG group the defect was filled with autologous BMC in combination with calcium phosphate granules (CPG, whereas in the BMC+CPG+PRP group the defect was filled with the composite of autologous BMC, CPG and autologous PRP. In the autograft group the defect was filled with autologous cancellous graft, whereas in the CPG group the defect was filled with CPG solely. After 6 weeks radiological and histomorphometrical analysis showed significantly more new bone formation in the BMC+CPG+PRP group compared to the BMC+CPG group and the CPG group. There were no significant differences between the BMC+CPG+PRP group and the autograft group. In the PRP platelets were enriched significantly about 4.7-fold compared to native blood. In BMC the count of mononuclear cells increased significantly (3.5-fold compared to the bone marrow aspirate. This study demonstrates that the composite of BMC+CPG+PRP leads to a significantly higher bone regeneration of critical-size defects at the proximal tibia in mini-pigs than the use of BMC+CPG without PRP. Furthermore, within the limits of the present study the composite BMC+CPG+PRP represents a comparable alternative to autologous bone grafting.

  12. What About the Rheological Properties of PRP/Microfat Mixtures in Fat Grafting Procedure?

    Science.gov (United States)

    Ghazouane, R; Bertrand, B; Philandrianos, C; Veran, J; Abellan, M; Francois, P; Velier, M; Orneto, C; Piccerelle, P; Magalon, J

    2017-10-01

    Fat grafting has emerged as a reference procedure in daily plastic surgery practice. Unpredictable fat resorption is the main clinical problem. For this purpose, the addition of PRP to enhance fat revascularization is now an easy and popular procedure. However, no consensus exists regarding the respective volume of fat and PRP used to obtain the ideal mixture. This study investigated the rheological properties of microfat mixed with different proportions of PRP. Results obtained were compared with commercialized hyaluronic acid fillers. Microfat and PRP preparations were performed using standardized techniques. Lipoaspirate residue and blood were obtained from six patients undergoing aesthetic facial microlipofilling. Elastic modulus G' and tan δ (proportion of elasticity versus fluidity) were obtained for the following conditions: microfat alone and microfat mixed with 10, 30 or 50% of PRP. An expected decrease in elastic modulus was observed by adding increase volumes of PRP. Two groups of products with different rheological properties were considered based on statistical differences highlighted regarding the value of G'. Mean tan δ varied from 0.20 ± 0.04 (microfat alone) to 0.28 ± 0.08 (50% microfat/50% PRP). Microfat mixed with 10% of PRP presents consistency comparable to stiffer fillers, whereas microfat mixed with 30 or 50% corresponds to softer fillers. Rheological differences were highlighted given the proportion of PRP added to the microfat. Further studies assessing the impact of increased doses of platelets in microfat/PRP mixtures on clinical outcomes should also be investigated. Our findings will help clinicians to choose a mixture that meets their specific needs for a given indication. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

  13. Increased susceptibility to Yersinia enterocolitica Infection of Tff2 deficient mice.

    Science.gov (United States)

    Shah, Aftab A; Mihalj, Martina; Ratkay, Ivana; Lubka-Pathak, Maria; Balogh, Peter; Klingel, Karin; Bohn, Erwin; Blin, Nikolaus; Baus-Loncar, Mirela

    2012-01-01

    TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica. Copyright © 2012 S. Karger AG, Basel.

  14. Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils.

    Science.gov (United States)

    Araman, Can; Thompson, Robert E; Wang, Siyao; Hackl, Stefanie; Payne, Richard J; Becker, Christian F W

    2017-09-01

    The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179-231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

  15. The differential effects of leukocyte-containing and pure platelet-rich plasma (PRP) on tendon stem/progenitor cells - implications of PRP application for the clinical treatment of tendon injuries.

    Science.gov (United States)

    Zhou, Yiqin; Zhang, Jianying; Wu, Haishan; Hogan, MaCalus V; Wang, James H-C

    2015-09-15

    Platelet-rich plasma (PRP) is widely used to treat tendon injuries in clinics. These PRP preparations often contain white blood cells or leukocytes, and the precise cellular effects of leukocyte-rich PRP (L-PRP) on tendons are not well defined. Therefore, in this study, we determined the effects of L-PRP on tendon stem/progenitor cells (TSCs), which play a key role in tendon homeostasis and repair. TSCs isolated from the patellar tendons of rabbits were treated with L-PRP or P-PRP (pure PRP without leukocytes) in vitro, followed by measuring cell proliferation, stem cell marker expression, inflammatory gene expression, and anabolic and catabolic protein expression by using immunostaining, quantitative real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay, respectively. Cell proliferation was induced by both L-PRP and P-PRP in a dose-dependent manner with maximum proliferation at a 10 % PRP dose. Both PRP treatments also induced differentiation of TSCs into active tenocytes. Nevertheless, the two types of PRP largely differed in several effects exerted on TSCs. L-PRP induced predominantly catabolic and inflammatory changes in differentiated tenocytes; its treatment increased the expression of catabolic marker genes, matrix metalloproteinase-1 (MMP-1), MMP-13, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α), and their respective protein expression and prostaglandin E2 (PGE 2) production. In contrast, P-PRP mainly induced anabolic changes; that is, P-PRP increased the gene expression of anabolic genes, alpha-smooth muscle actin (α-SMA), collagen types I and III. These findings indicate that, while both L-PRP and P-PRP appear to be "safe" in inducing TSC differentiation into active tenocytes, L-PRP may be detrimental to the healing of injured tendons because it induces catabolic and inflammatory effects on tendon cells and may prolong the effects in healing tendons. On the other hand, when P-PRP is used to

  16. A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles

    Science.gov (United States)

    Mercer, Robert C. C.; Fu, Ze-Lin; Mays, Charles E.; Gapeshina, Hristina; Wohlgemuth, Serene L.; Acevedo-Morantes, Claudia Y.; Cashman, Neil R.; Coulthart, Michael B.; Jansen, Gerard H.; Stepanova, Maria

    2018-01-01

    To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting de novo formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie. PMID:29338055

  17. Effects of platelet rich plasma (PRP) on human gingival fibroblast, osteoblast and periodontal ligament cell behaviour.

    Science.gov (United States)

    Kobayashi, Eizaburo; Fujioka-Kobayashi, Masako; Sculean, Anton; Chappuis, Vivianne; Buser, Daniel; Schaller, Benoit; Dőri, Ferenc; Miron, Richard J

    2017-06-02

    The use of platelet rich plasma (PRP, GLO) has been used as an adjunct to various regenerative dental procedures. The aim of the present study was to characterize the influence of PRP on human gingival fibroblasts, periodontal ligament (PDL) cells and osteoblast cell behavior in vitro. Human gingival fibroblasts, PDL cells and osteoblasts were cultured with conditioned media from PRP and investigated for cell migration, proliferation and collagen1 (COL1) immunostaining. Furthermore, gingival fibroblasts were tested for genes encoding TGF-β, PDGF and COL1a whereas PDL cells and osteoblasts were additionally tested for alkaline phosphatase (ALP) activity, alizarin red staining and mRNA levels of osteoblast differentiation markers including Runx2, COL1a2, ALP and osteocalcin (OCN). It was first found that PRP significantly increased cell migration of all cells up to 4 fold. Furthermore, PRP increased cell proliferation at 3 and 5 days of gingival fibroblasts, and at 3 days for PDL cells, whereas no effect was observed on osteoblasts. Gingival fibroblasts cultured with PRP increased TGF-β, PDGF-B and COL1 mRNA levels at 7 days and further increased over 3-fold COL1 staining at 14 days. PDL cells cultured with PRP increased Runx2 mRNA levels but significantly down-regulated OCN mRNA levels at 3 days. No differences in COL1 staining or ALP staining were observed in PDL cells. Furthermore, PRP decreased mineralization of PDL cells at 14 days post seeding as assessed by alizarin red staining. In osteoblasts, PRP increased COL1 staining at 14 days, increased COL1 and ALP at 3 days, as well as increased ALP staining at 14 days. No significant differences were observed for alizarin red staining of osteoblasts following culture with PRP. The results demonstrate that PRP promoted gingival fibroblast migration, proliferation and mRNA expression of pro-wound healing molecules. While PRP induced PDL cells and osteoblast migration and proliferation, it tended to have

  18. Comparison of the Efficacy of Homologous and Autologous Platelet-Rich Plasma (PRP) for Treating Androgenic Alopecia.

    Science.gov (United States)

    Ince, Bilsev; Yildirim, Mehmet Emin Cem; Dadaci, Mehmet; Avunduk, Mustafa Cihat; Savaci, Nedim

    2018-02-01

    Androgenetic alopecia (AGA), the most common cause of hair loss in both sexes, accounts for 95% of all cases of hair loss. Although the literature has suggested that both nonactivated (n-PRP) and activated autologous (a-PRP) PRP can be used to treat AGA, we did not find any study investigating the use of homologous PRP (h-PRP) for this purpose. Also, to the best of our knowledge, there are no studies comparing the efficacy of h-PRP, a-PRP, or n-PRP on AGA therapy. The aim of this study was to compare the increase in hair density, average number of platelets, complications, preparation, and duration of application in the treatment of AGA using a-PRP, n-PRP, and h-PRP. Between 2014 and 2015, we studied male patients who had experienced increased hair loss in the last year. Patients were divided into three groups: Group 1 received n-PRP, Group 2 received active PRP, and Group 3 received h-PRP. For Group 1, PRP was prepared by a single centrifugation prepared from the patient's own blood. For Group 2, the PRP was prepared from the patient's own blood, but a second centrifugation was applied for platelet activation with calcium chloride. For Group 3, the PRP was prepared from pooled platelets with the same blood group as the patient from the blood center. PRP was injected at 1, 2, and 6 months. The hair density (n/cm 2 ) of each patient before and after injection was calculated. Each patient was assigned a fixed evaluation point at the time of application to calculate hair density. At 2, 6, and 12 months after the first treatment, the increase in hair density was calculated as 11.2, 26.1, and 32.4%, respectively, in Group 1; 8.1, 12.5, and 20.8%, respectively, in Group 2; and 16.09, 36.41, and 41.76%, respectively, in Group 3. The increase in hair density was statistically significantly greater in Group 1 than in Group 2 and more so in Group 3 than in both groups among all controls (p PRP was greater than with autologous PRP groups. We believe that h-PRP therapy can

  19. PrP N-terminal domain triggers PrPSc-like aggregation of Dpl

    International Nuclear Information System (INIS)

    Erlich, Paul; Cesbron, Jean-Yves; Lemaire-Vieille, Catherine; Curt, Aurelie; Andrieu, Jean-Pierre; Schoehn, Guy; Jamin, Marc; Gagnon, Jean

    2008-01-01

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP C , for 'cellular prion protein') into an abnormal state (PrP Sc , for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP C . In contrast to its homologue PrP C , Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP Sc -like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy

  20. The antimicrobial peptide cathelicidin protects mice from Escherichia coli O157:H7-mediated disease.

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    Milan Chromek

    Full Text Available This study investigated the role of the antimicrobial peptide cathelicidin in Escherichia coli O157:H7 infection and subsequent renal damage. Mouse and human cathelicidin, CRAMP and LL-37, respectively, killed E. coli O157:H7 in vitro. Intestines from healthy wild-type (129/SvJ and cathelicidin-knock-out (Camp(-/- mice were investigated, showing that cathelicidin-deficient mice had a thinner colonic mucus layer compared with wild-type mice. Wild-type (n = 11 and cathelicidin-knock-out (n = 11 mice were inoculated with E. coli O157:H7. Cathelicidin-deficient animals exhibited higher fecal counts of E. coli O157:H7 and bacteria penetrated the mucus forming attaching-and-effacing lesions to a much higher extent than in wild-type animals. Cathelicidin knock-out mice developed symptoms (9/11 as well as anemia, thrombocytopenia and extensive renal tubular damage while all cathelicidin-producing mice remained asymptomatic with normal laboratory findings. When injected with Shiga toxin intraperitoneally, both murine strains developed the same degree of renal tubular damage and clinical disease indicating that differences in sensitivity to infection between the murine strains were related to the initial intestinal response. In conclusion, cathelicidin substantially influenced the antimicrobial barrier in the mouse colon mucosa. Cathelicidin deficiency lead to increased susceptibility to E. coli O157:H7 infection and subsequent renal damage. Administration of cathelicidin or stimulation of endogenous production may prove to be novel treatments for E. coli O157:H7-induced hemolytic uremic syndrome.

  1. PRP for Degenerative Cartilage Disease: A Systematic Review of Clinical Studies.

    Science.gov (United States)

    Laver, Lior; Marom, Niv; Dnyanesh, Lad; Mei-Dan, Omer; Espregueira-Mendes, João; Gobbi, Alberto

    2017-10-01

    To explore the utilization of platelet-rich plasma (PRP) for degenerative cartilage processes and evaluate whether there is sufficient evidence to better define its potential effects. Systematic literature reviews were conducted in PubMed/MEDLINE and Cochrane electronic databases till May 2015, using the keywords "platelet-rich plasma OR PRP OR autologous conditioned plasma OR ACP AND cartilage OR chondrocyte OR chondrogenesis OR osteoarthritis (OA) OR arthritis." The final result yielded 29 articles. Twenty-six studies examined PRP administration for knee OA and 3 involved PRP administration for hip OA. The results included 9 prospective randomized controlled trials (RCTs) (8 knee and 1 hip), 4 prospective comparative studies, 14 case series, and 2 retrospective comparative studies. Hyaluronic acid (HA) was used as a control in 11 studies (7 RCTs, 2 prospective comparative studies, and 2 retrospective cohort). Overall, all RCTs reported on improved symptoms compared to baseline scores. Only 2 RCTs-one for knee and one for hip-did not report significant superiority of PRP compared to the control group (HA). Nine out of 11 HA controlled studies showed significant better results in the PRP groups. A trend toward better results for PRP injections in patients with early knee OA and young age was observed; however, lack of uniformity was evident in terms of indications, inclusion criteria, and pathology definitions in the different studies. Current clinical evidence supports the benefit in PRP treatment for knee and hip OA, proven to temporarily relieve pain and improve function of the involved joint with superior results compared with several alternative treatments. Further research to establish the optimal preparation protocol and characteristics of PRP injections for OA is needed.

  2. Kartogenin with PRP promotes the formation of fibrocartilage zone in the tendon-bone interface.

    Science.gov (United States)

    Zhou, Yiqin; Zhang, Jianying; Yang, Jinsong; Narava, Manoj; Zhao, Guangyi; Yuan, Ting; Wu, Haishan; Zheng, Nigel; Hogan, MaCalus V; Wang, James H-C

    2017-12-01

    Treatment of tendon-bone junction injuries is a challenge because tendon-bone interface often heals poorly and the fibrocartilage zone, which reduces stress concentration, at the interface is not formed. In this study, we used a compound called kartogenin (KGN) with platelet-rich plasma (PRP) to induce the formation of fibrocartilage zone in a rat tendon graft-bone tunnel model. The experimental rats received KGN-PRP or PRP injections in the tendon graft-bone tunnel interface. The control group received saline. After 4, 8 and 12 weeks, Safranin O staining of the tendon graft-bone tunnels revealed abundant proteoglycans in the KGN-PRP group indicating the formation of cartilage-like transition zone. Immunohistochemical and immuno-fluorescence staining revealed collagen types I (Col-I) and II (Col-II) in the newly formed fibrocartilage zone. Both fibrocartilage zone formation and maturation were healing time dependent. In contrast, the PRP and saline control groups had no cartilage-like tissues and minimal Col-I and Col-II staining. Some gaps were also present in the saline control group. Finally, pull-out strength in the KGN-PRP-treated group at 8 weeks was 1.4-fold higher than the PRP-treated group and 1.6-fold higher than the saline control group. These findings indicate that KGN, with PRP as a carrier, promotes the formation of fibrocartilage zone between the tendon graft and bone interface. Thus, KGN-PRP may be used as a convenient cell-free therapy in clinics to promote fibrocartilage zone formation in rotator calf repair and anterior cruciate ligament reconstruction, thereby enhancing the mechanical strength of the tendon-bone interface and hence the clinical outcome of these procedures. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  3. CD36 participates in PrP(106-126-induced activation of microglia.

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    Mohammed Kouadir

    Full Text Available Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP fragment 106-126 (PrP(106-126. We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126 in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb. The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126. The results showed that PrP(106-126 treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α, increased iNOS expression and nitric oxide (NO production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126. Together, these results suggest that CD36 is involved in PrP(106-126-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126 and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides

  4. Singlet ground-state fluctuations in praseodymium observed by muon spin relaxation in PrP and PrP0.9

    International Nuclear Information System (INIS)

    Noakes, D R; Waeppling, R; Kalvius, G M; Jr, M F White; Stronach, C E

    2005-01-01

    Muon spin relaxation (μSR) in the singlet ground-state compounds PrP and PrP 0.9 reveals the unusual situation of a Lorentzian local field distribution with fast-fluctuation-limit strong-collision dynamics, a case that does not show motional narrowing. Contrary to publications by others, where PrP 0.9 was asserted to have vacancy-induced spin-glass freezing, no spin-glass freezing is seen in PrP 0.9 or PrP down to ≤100mK. This was confirmed by magnetization measurements on these same samples. In both compounds, the muon spin relaxation rate does increase as temperature decreases, demonstrating increasing strength of the paramagnetic response. A Monte Carlo model of fluctuations of Pr ions out of their crystalline-electric-field singlet ground states into their magnetic excited states (and back down again) produces the strong-collision-dynamic Lorentzian relaxation functions observed at each individual temperature but not the observed temperature dependence. This model contains no exchange interaction, and so predicts decreasing paramagnetic response as the temperature decreases, contrary to the temperature dependence observed. Comparison of the simulations to the data suggests that the exchange interaction is causing the system to approach magnetic freezing (by mode softening), but fails to complete the process

  5. A call for a standard classification system for future biologic research: the rationale for new PRP nomenclature.

    Science.gov (United States)

    Mautner, Kenneth; Malanga, Gerard A; Smith, Jay; Shiple, Brian; Ibrahim, Victor; Sampson, Steven; Bowen, Jay E

    2015-04-01

    Autologous cell therapies including platelet-rich plasma (PRP) and bone marrow concentrate (BMC) are increasingly popular options for soft tissue and joint-related diseases. Despite increased clinical application, conflicting research has been published regarding the efficacy of PRP, and few clinical publications pertaining to BMC are available. Preparations of PRP (and BMC) can vary in many areas, including platelet concentration, number of white blood cells, presence or absence of red blood cells, and activation status of the preparation. The potential effect of PRP characteristics on PRP efficacy is often not well understood by the treating clinician, and PRP characteristics, as well as the volume of PRP delivered, are unfortunately not included in the methods of many published research articles. It is essential to establish a standard reporting system for PRP that facilitates communication and the interpretation and synthesis of scientific investigations. Herein, the authors propose a new PRP classification system reflecting important PRP characteristics based on contemporary literature and recommend adoption of minimal standards for PRP reporting in scientific investigations. Widespread adoption of these recommendations will facilitate interpretation and comparison of clinical studies and promote scientifically based progress in the field of regenerative medicine. Copyright © 2015 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.

  6. Regulatory effects of adenosine A2A receptors on psychomotor ability and mood behavior of mice

    Directory of Open Access Journals (Sweden)

    Li JIANG

    2011-07-01

    Full Text Available Objective To explore the effects of gene knock-out,agonist or inhibitor of adenosine A2A receptor on the locomotor activity,and anxiety-or depression-like behavior of mice.Methods Male C57BL/6 mice,comprising those underwent gene knock-out of adenosine A2A receptor(A2AKO and their wild-type(WT littermates,were assigned into A2AKO group and WT group.Another batch of male C57BL/6,specific-pathogen-free(SPF mice,were assigned into SCH58261 group,CGS21680 group and control group.Mice of aforesaid 3 groups were transperitoneally administered with SCH58261,a specific inhibitor of adenosine A2A receptor at a dose of 2mg/kg,CGS21680,a specific agonist of adenosine A2A receptor at a dose of 0.5mg/kg,and vehicle(0.25ml,comprising DMSO and saline,respectively.Ten minutes after injection,mice of the 3 groups underwent open-field test,elevated plus-maze test and forced swimming test to detect their locomotor activity,anxiety-and depression-like behavior.Results a Compared with WT group,the total movement distance decreased(P 0.05.b Compared with control group,the total movement distance decreased and the stay time in the peripheral area increased significantly in the open field test(P 0.05.Conclusions The agonist of adenosine A2A receptor may depress the spontaneous motility and exploratory behavior,and exacerbate the anxiety and depression,and it simulates the effect induced by knock-out of A2A receptor gene,but it is opposite to the effect induced by A2A receptor inhibitor.

  7. Comparison of hyaluronic acid and PRP intra-articular injection with combined intra-articular and intraosseous PRP injections to treat patients with knee osteoarthritis.

    Science.gov (United States)

    Su, Ke; Bai, Yuming; Wang, Jun; Zhang, Haisen; Liu, Hao; Ma, Shiyun

    2018-05-01

    The aim of this study was to evaluate the benefit provided by intraosseous infiltration combined with intra-articular injection of platelet-rich plasma to treat mild and moderate stages of knee joint degeneration (Kellgren-Lawrence score II-III) compared with other treatments, specifically intra-articular injection of PRP and of HA. Eighty-six patients with grade II to grade III knee OA according to the Kellgren-Lawrence classification were randomly assigned to intra-articular combined with intraosseous injection of PRP (group A), intra-articular PRP (group B), or intra-articular HA (group C). Patients in group A received intra-articular combined with intraosseous injection of PRP (administered twice, 2 weeks apart). Patients in group B received intra-articular injection of PRP every 14 days. Patients in group C received a series of five intra-articular injections of HA every 7 days. All patients were evaluated using the Visual Analogue Scale (VAS) and Western Ontario and McMaster Universities (WOMAC) score before the treatment and at 1, 3, 6, 12, and 18 months after treatment. There were significant improvements at the end of the 1st month. Notably, group A patients had significantly superior VAS and WOMAC scores than were observed in groups B and C. The VAS scores were similar in groups B and group C after the 6th month. Regarding the WOMAC scores, groups B and C differed at the 1st, 3rd, 6th, and 12th months; however, no significant difference was observed at the 18th month. The combination of intraosseous with intra-articular injections of PRP resulted in a significantly superior clinical outcome, with sustained lower VAS and WOMAC scores and improvement in quality of life within 18 months.

  8. Experimental conditions and monitoring items of the prototype repository project (PRP). Research document

    International Nuclear Information System (INIS)

    Sugita, Yutaka; Ito, Akira; Kawakami, Susumu

    2003-03-01

    Various experiments are ongoing in the underground research facility 'the Hard Rock Laboratory (HRL)' of SKB in Sweden for the geological disposal of the high-level radioactive waste. International joint project Prototype Repository Project (PRP) is one of the experiments in the HRL which has some engineered barrier systems and to study the coupled behavior happening in and around the engineered barrier system. JNC has joined this international joint project PRP to obtain the information of the coupled behavior on such systematic engineered barrier system and to apply the JNC's coupled THMC analytical code to the prediction and back analysis of the PRP. The analytical code will be verified through these analyses in this project. JNC can apply the verified analytical code to assess the coupled behavior in Japan. This report summarizes the experimental conditions and monitoring items of the PRP. (author)

  9. Role of Ultrasound Guided Platelet-Rich Plasma (PRP Injection in Treatment of Lateral Epicondylitis

    Directory of Open Access Journals (Sweden)

    Enass M. Khattab

    2017-06-01

    Conclusion: We concluded that US-guided platelet-rich plasma (PRP injection for treatment of lateral epicondylitis was a safe, minimally invasive and effective procedure in improving the sonographic and pathological changes of common extensor tendon (CET.

  10. Lumbar Intradiskal Platelet-Rich Plasma (PRP) Injections: A Prospective, Double-Blind, Randomized Controlled Study.

    Science.gov (United States)

    Tuakli-Wosornu, Yetsa A; Terry, Alon; Boachie-Adjei, Kwadwo; Harrison, Julian R; Gribbin, Caitlin K; LaSalle, Elizabeth E; Nguyen, Joseph T; Solomon, Jennifer L; Lutz, Gregory E

    2016-01-01

    To determine whether single injections of autologous platelet-rich plasma (PRP) into symptomatic degenerative intervertebral disks will improve participant-reported pain and function. Prospective, double-blind, randomized controlled study. Outpatient physiatric spine practice. Adults with chronic (≥6 months), moderate-to-severe lumbar diskogenic pain that was unresponsive to conservative treatment. Participants were randomized to receive intradiskal PRP or contrast agent after provocative diskography. Data on pain, physical function, and participant satisfaction were collected at 1 week, 4 weeks, 8 weeks, 6 months, and 1 year. Participants in the control group who did not improve at 8 weeks were offered the option to receive PRP and subsequently followed. Functional Rating Index (FRI), Numeric Rating Scale (NRS) for pain, the pain and physical function domains of the 36-item Short Form Health Survey, and the modified North American Spine Society (NASS) Outcome Questionnaire were used. Forty-seven participants (29 in the treatment group, 18 in the control group) were analyzed by an independent observer with a 92% follow-up rate. Over 8 weeks of follow-up, there were statistically significant improvements in participants who received intradiskal PRP with regards to pain (NRS Best Pain) (P = .02), function (FRI) (P = .03), and patient satisfaction (NASS Outcome Questionnaire) (P = .01) compared with controls. No adverse events of disk space infection, neurologic injury, or progressive herniation were reported following the injection of PRP. Participants who received intradiskal PRP showed significant improvements in FRI, NRS Best Pain, and NASS patient satisfaction scores over 8 weeks compared with controls. Those who received PRP maintained significant improvements in FRI scores through at least 1 year of follow-up. Although these results are promising, further studies are needed to define the subset of participants most likely to respond to biologic intradiskal

  11. DEPA classification: a proposal for standardising PRP use and a retrospective application of available devices.

    Science.gov (United States)

    Magalon, J; Chateau, A L; Bertrand, B; Louis, M L; Silvestre, A; Giraudo, L; Veran, J; Sabatier, F

    2016-01-01

    Significant biological differences in platelet-rich plasma (PRP) preparations have been highlighted and could explain the large variability in the clinical benefit of PRP reported in the literature. The scientific community now recommends the use of classification for PRP injection; however, these classifications are focused on platelet and leucocyte concentrations. This presents the disadvantages of (1) not taking into account the final volume of the preparation; (2) omitting the presence of red blood cells in PRP and (3) not assessing the efficiency of production. On the basis of standards classically used in the Cell Therapy field, we propose the DEPA (Dose of injected platelets, Efficiency of production, Purity of the PRP, Activation of the PRP) classification to extend the characterisation of the injected PRP preparation. We retrospectively applied this classification on 20 PRP preparations for which biological characteristics were available in the literature. Dose of injected platelets varies from 0.21 to 5.43 billion, corresponding to a 25-fold increase. Only a Magellan device was able to obtain an A score for this parameter. Assessments of the efficiency of production reveal that no device is able to recover more than 90% of platelets from the blood. Purity of the preparation reveals that a majority of the preparations are contaminated by red blood cells as only three devices reach an A score for this parameter, corresponding to a percentage of platelets compared with red blood cells and leucocytes over 90%. These findings should provide significant help to clinicians in selecting a system that meets their specific needs for a given indication.

  12. Platelet-rich plasma (PRP) treatment of sports-related severe acute hamstring injuries.

    Science.gov (United States)

    Guillodo, Yannick; Madouas, Gwénaelle; Simon, Thomas; Le Dauphin, Hermine; Saraux, Alain

    2015-01-01

    hamstring injury is the most common musculoskeletal disorder and one of the main causes of missed sporting events. Shortening the time to return to play (TTRTP) is a priority for athletes and sports medicine practitioners. platelet-rich plasma (PRP) injection at the site of severe acute hamstring injury increases the healing rate and shortens the TTRTP. Cohort study. all patients with ultrasonography and MRI evidence of severe acute hamstring injury between January 2012 and March 2014 were offered PRP treatment. Those who accepted received a single intramuscular PRP injection within 8 days post-injury; the other patients served as controls. The same standardized rehabilitation program was used in both groups. A physical examination and ultrasonography were performed 10 and 30 days post-injury, then a phone interview 120 days post-injury, to determine the TTRTP at the pre-injury level. of 34 patients, 15 received PRP and 19 did not. Mean TTRTP at the pre-injury level was 50.9±10.7 days in the PRP group and 52.8±15.7 days in the control group. The difference was not statistically significant. a single intramuscular PRP injection did not shorten the TTRTP in sports people with severe acute hamstring injuries.

  13. Effectiveness of PRP Injection in Reducing Recovery Time of Acute Hamstring Injury: A Critically Appraised Topic.

    Science.gov (United States)

    Manduca, Mary Lynn; Straub, Stephen J

    2017-07-17

    Clinical Scenario Hamstring strains are common athletic injuries, with a high recurrence rate (34%). 2 Recently, platelet-rich-plasma (PRP) injections have gained popularity as a potential treatment option to accelerate healing of hamstring injury. 3 Focused Clinical Question Does the combination of PRP injection and rehabilitation decrease recovery time of acute hamstring injury as compared to rehabilitation alone in collegiate athletes? Summary of Key Findings A literature search resulted in three randomized controlled trials (RCT). One study showed benefits in various outcome measures with PRP, compared to rehabilitation alone, while two showed no benefits. One study reported improved pain, ultrasonography regenerative indications, and recovery time with PRP injection following acute hamstring injury 1 , however, larger studies have shown no benefits. 7-9 The literature demonstrates conflicting evidence regarding benefits of PRP injections in hamstring injuries. Clinical Bottom Line At this time, PRP injections cannot be recommended as having value for hamstring injuries, compared to rehabilitation alone. Strength of Recommendation Due to inconsistent or limited quality patient-oriented evidence in existing literature, the strength of this recommendation is grade B, based on the Strength of Recommendation Taxonomy (SORT). 7 .

  14. Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

    Science.gov (United States)

    Guizzunti, Gianni; Zurzolo, Chiara

    2015-01-01

    We previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death.

  15. Polymorphisms at Amino Acid Residues 141 and 154 Influence Conformational Variation in Ovine PrP

    Science.gov (United States)

    Yang, Sujeong; Thackray, Alana M.; Hopkins, Lee; Monie, Tom P.; Burke, David F.; Bujdoso, Raymond

    2014-01-01

    Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie. PMID:25126555

  16. Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia.

    Science.gov (United States)

    Gentile, Pietro; Garcovich, Simone; Scioli, Maria Giovanna; Bielli, Alessandra; Orlandi, Augusto; Cervelli, Valerio

    2018-01-27

    23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected. The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm 2 ) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed. PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

  17. Mouse homologue of yeast Prp19 interacts with mouse SUG1, the regulatory subunit of 26S proteasome

    International Nuclear Information System (INIS)

    Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon

    2007-01-01

    Yeast Prp19 has been shown to involve in pre-mRNA splicing and DNA repair as well as being an ubiquitin ligase. Mammalian homologue of yeast Prp19 also plays on similar functional activities in cells. In the present study, we isolated mouse SUG1 (mSUG1) as binding partner of mouse Prp19 (mPrp19) by the yeast two-hybrid system. We confirmed the interaction of mPrp9 with mSUG1 by GST pull-down assay and co-immunoprecipitation assay. The N-terminus of mPrp19 including U-box domain was associated with the C-terminus of mSUG1. Although, mSUG1 is a regulatory subunit of 26S proteasome, mPrp19 was not degraded in the proteasome-dependent pathway. Interestingly, GFP-mPrp19 fusion protein was co-localized with mSUG1 protein in cytoplasm as the formation of the speckle-like structures in the presence of a proteasome inhibitor MG132. In addition, the activity of proteasome was increased in cells transfected with mPrp19. Taken together, these results suggest that mPrp19 involves the regulation of protein turnover and may transport its substrates to 26S proteasome through mSUG1 protein

  18. Extraneural manifestations of prion infection in GPI-anchorless transgenic mice

    International Nuclear Information System (INIS)

    Lee, Andrew M.; Paulsson, Johan F.; Cruite, Justin; Andaya, Abegail A.; Trifilo, Matthew J.; Oldstone, Michael B.A.

    2011-01-01

    Earlier studies indicated that transgenic (tg) mice engineered to express prion protein (PrP) lacking the glycophosphatidylinositol (GPI -/- ) membrane anchor formed abnormal proteinase-resistant prion (PrPsc) amyloid deposits in their brains and hearts when infected with the RML strain of murine scrapie. In contrast, RML scrapie infection of normal mice with a GPI-anchored PrP did not deposit amyloid with PrPsc in the brain or the heart. Here we report that scrapie-infected GPI -/- PrP tg mice also deposit PrP and transmissible infectious material in the gut, kidneys, and islets of Langerhans. Similar to previously reported amyloid deposits in the brain and heart, amyloid deposits were found in the gut; however, no amyloid deposited in the islets. By high-resolution electron microscopy, we show PrP is located primarily in α cells and also β cells. Islets contain abundant insulin and there is no abnormality in glucose metabolism in infected GPI -/- PrP tg mice.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  20. Transgenic fatal familial insomnia mice indicate prion infectivity-independent mechanisms of pathogenesis and phenotypic expression of disease.

    Directory of Open Access Journals (Sweden)

    Ihssane Bouybayoune

    2015-04-01

    Full Text Available Fatal familial insomnia (FFI and a genetic form of Creutzfeldt-Jakob disease (CJD178 are clinically different prion disorders linked to the D178N prion protein (PrP mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD mice modeling CJD178. No prion infectivity was detectable in Tg(FFI and Tg(CJD brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI and Tg(CJD neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.

  1. Exercise and the platelet activator calcium chloride both influence the growth factor content of platelet-rich plasma (PRP): overlooked biochemical factors that could influence PRP treatment

    NARCIS (Netherlands)

    Hamilton, Bruce; Tol, Johannes L.; Knez, Wade; Chalabi, Hakim

    2015-01-01

    There is strong evidence that exercise affects platelet haemostasis factors, but this potential effect on growth factor concentrations in platelet-rich plasma (PRP) has never been studied. In addition, there is a paucity of studies focusing on the effects of activating agents used in conjunction

  2. The influence of using anticoagulants (EDTA and citrate acid 3.8% toward the quantity of Platelet Rich Plasma (PRP

    Directory of Open Access Journals (Sweden)

    Lilies Anggarwati Astuti

    2016-06-01

    Full Text Available Platelet Rich Plasma (PRP is a blood concentrate that has a thrombocytes concentration several time higher than normal concentration of thrombocytes in normal human blood. PRP is a promising alternative to surgery with a safe and natural healing. The standard protocol for PRP preparation must be determined to get the right quantity and quality of the matrix of fibrin, leukocytes, platelets and growth factors. It could not be separated from the number of PRP produced. The use of PRP in the success of periodontal treatment would not be separated from methods to obtain it. To detect the influence of using anticoagulants (EDTA and citrate acid 3.8% toward the quantity of PRP. There are 41 subjects studied by taking 21 ml of venous blood in each of the seven tubes. Centrifugation performed twice with different speed, duration, use of anticoagulants then analyzed. This quantity between the two groups differed significantly between the PRP in EDTA group is higher 322.2 ml rather than citrate acid 3.8% group, then control group is higher 329.5 ml rather than citrate acid 3.8% group, while there is no difference between EDTA and control group. There is effect of the use of anticoagulants EDTA compared with citrate acid 3.8% in the quantity of PRP, and there was no effect using citrate acid 3.8% as anticoagulants in quantity of PRP.

  3. Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy

    Science.gov (United States)

    Cortes, Constanza J.; Qin, Kefeng; Norstrom, Eric M.; Green, William N.; Bindokas, Vytautas P.; Mastrianni, James A.

    2013-01-01

    Prion diseases are linked to the accumulation of a misfolded isoform (PrPSc) of prion protein (PrP). Evidence suggests that lysosomes are degradation endpoints and sites of the accumulation of PrPSc. We questioned whether lysosomes participate in the early quality control of newly generated misfolded PrP. We found PrP carrying the disease-associated T182A mutation (Mut-PrP) was delivered to lysosomes in a Golgi-independent manner. Time-lapse live cell imaging revealed early formation and uptake of GFP-tagged Mut-PrP aggregates into LysoTracker labeled vesicles. Compared with Wt-PrP, Mut-PrP expression was associated with an elevation in several markers of the autophagy-lysosomal pathway, and it extensively colocalized with the autophagosome-specific marker, LC3B. In autophagy deficient (ATG5−/−) mouse embryonic fibroblasts, or in normal cells treated with the autophagy-inhibitor 3-MA, Mut-PrP colocalization with lysosomes was reduced to a similar extent. Additionally, 3-MA selectively impaired the degradation of insoluble Mut-PrP, resulting in an increase in protease-resistant PrP, whereas the induction of autophagy by rapamycin reduced it. These findings suggest that autophagy might function as a quality control mechanism to limit the accumulation of misfolded PrP that normally leads to the generation of PrPSc. PMID:24454378

  4. Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein.

    Science.gov (United States)

    Watts, Joel C; Giles, Kurt; Stöhr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K; Patel, Smita; DeArmond, Stephen J; Prusiner, Stanley B

    2012-02-28

    Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.

  5. Successful treatment of radiation retinopathy with panretinal photocoagulation (PRP) in a patient of orbital MALT lymphoma

    International Nuclear Information System (INIS)

    Okamoto, Hiroyuki; Chikuda, Makoto; Kadoya, Kouji

    2012-01-01

    Report a case of satisfactory progress radiation retinopathy after radiation for mucosa-associated lymphoid tissue (MALT) lymphoma. A 26-year-old male patient, referred to our department for lacrimal sac tumor. Biopsy was done by otolaryngology and radiation therapy was performed (total irradiation of 41.4 Gy) as pathological examination revealed MALT lymphoma. Soft exudates and macula edema appeared in posterior pole of the right fundus after radiotherapy. Right vision became 0.5 because of macula edema, and panretinal photocoagulation (PRP) was performed. After PRP, macula edema withdrew and right vision improved to 1.2. It is suggested that the fundus must be monitored after radiation therapy, and early treatment, such as PRP is effective in radiation retinopathy. (author)

  6. Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils.

    Science.gov (United States)

    Twomey, Megan C; Brooks, Jenna K; Corey, Jillaine M; Singh-Cundy, Anu

    2013-10-15

    An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins). Copyright © 2013 Elsevier GmbH. All rights reserved.

  7. Effect of Use of Platelet-Rich Plasma (PRP) in Skin with Intrinsic Aging Process.

    Science.gov (United States)

    Charles-de-Sá, Luiz; Gontijo-de-Amorim, Natale Ferreira; Takiya, Christina Maeda; Borojevic, Radovan; Benati, Donatella; Bernardi, Paolo; Sbarbati, Andrea; Rigotti, Gino

    2018-02-15

    In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous component. The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results. This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injection was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed. Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

  8. Genetic human prion disease modelled in PrP transgenic Drosophila.

    Science.gov (United States)

    Thackray, Alana M; Cardova, Alzbeta; Wolf, Hanna; Pradl, Lydia; Vorberg, Ina; Jackson, Walker S; Bujdoso, Raymond

    2017-09-20

    Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrP Sc , an abnormal isomer of the normal host protein PrP C , in the brain of affected individuals. PrP Sc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host. © 2017 The Author(s).

  9. Recombinant PrP and Its Contribution to Research on Transmissible Spongiform Encephalopathies.

    Science.gov (United States)

    Charco, Jorge M; Eraña, Hasier; Venegas, Vanessa; García-Martínez, Sandra; López-Moreno, Rafael; González-Miranda, Ezequiel; Pérez-Castro, Miguel Ángel; Castilla, Joaquín

    2017-12-14

    The misfolding of the cellular prion protein (PrP C ) into the disease-associated isoform (PrP Sc ) and its accumulation as amyloid fibrils in the central nervous system is one of the central events in transmissible spongiform encephalopathies (TSEs). Due to the proteinaceous nature of the causal agent the molecular mechanisms of misfolding, interspecies transmission, neurotoxicity and strain phenomenon remain mostly ill-defined or unknown. Significant advances were made using in vivo and in cellula models, but the limitations of these, primarily due to their inherent complexity and the small amounts of PrP Sc that can be obtained, gave rise to the necessity of new model systems. The production of recombinant PrP using E. coli and subsequent induction of misfolding to the aberrant isoform using different techniques paved the way for the development of cell-free systems that complement the previous models. The generation of the first infectious recombinant prion proteins with identical properties of brain-derived PrP Sc increased the value of cell-free systems for research on TSEs. The versatility and ease of implementation of these models have made them invaluable for the study of the molecular mechanisms of prion formation and propagation, and have enabled improvements in diagnosis, high-throughput screening of putative anti-prion compounds and the design of novel therapeutic strategies. Here, we provide an overview of the resultant advances in the prion field due to the development of recombinant PrP and its use in cell-free systems.

  10. Distribution, recovery and concentration of platelets and leukocytes in L-PRP prepared by centrifugation.

    Science.gov (United States)

    de Melo, Bruna Alice Gomes; Martins Shimojo, Andréa Arruda; Marcelino Perez, Amanda Gomes; Duarte Lana, José Fabio Santos; Andrade Santana, Maria Helena

    2018-01-01

    Platelet-rich plasma (PRP) is an autologous product prepared from whole blood (WB) that is widely used in regenerative medicine. In clinical practice, discontinuous centrifugation is used for both hand- and machine-prepared PRP. However, separation of WB fractions via centrifugation is a complex process, and the lack of clear mechanisms limits the understanding and evaluation of PRP preparation methods This paper focuses on the distribution, recovery and concentration factor of platelets and leukocytes in L-PRP (leukocyte and platelet-rich plasma) to define a concentration pattern for these blood components due to centrifugation conditions. WB collected from three healthy donors was centrifuged for 10min at 50-800 xg in a first step and then at 400 xg in a second step. The results from the first centrifugation step showed most platelets to be distributed in the upper layer (UL) and the buffy coat (BC), with approximately 14.5±5.2% retained in the bottom layer (BL). Most leukocytes were present in the BL. The greatest platelet recoveries from L-PRP were obtained at up to 150 xg (88.5±16.9%). The cumulative concentration factors with respect to the WB from the second centrifugation step were 6 and 1.2 for platelets and leukocytes, respectively. Thus, the concentration patterns delineated three centrifugation ranges with platelet/leukocyte ratios of 205±18, 325±15 and 107±4 and lymphocyte/granulocyte ratios of 1.54±0.74, 0.90±0.08 and 0.42±0.07. These findings contribute to a scientifically based standardization of L-PRP preparations. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Site enforcement tracking system (SETS): PRP listing by site for region 9

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  12. Site enforcement tracking system (SETS): PRP listing by site for region 8

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  13. Site enforcement tracking system (SETS): PRP listing by site for region 10

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  14. Site enforcement tracking system (SETS): PRP listing by site for region 3

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  15. Site enforcement tracking system (SETS): PRP listing by site for region 2

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  16. Site enforcement tracking system (SETS): PRP listing by site for region 5

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  17. Site enforcement tracking system (SETS): PRP listing by site for region 6

    International Nuclear Information System (INIS)

    1993-04-01

    When expending Superfund monies at a CERCLA (Comprehensive Environmental Response, Compensation and Liability Act) site, EPA must conduct a search to identify parties with potential financial responsibility for remediation of uncontrolled hazardous waste sites. EPA regional Superfund Waste Management Staff issue a notice letter to the potentially responsible party (PRP). Data from the notice letter is used to form the Site Enforcement Tracking System (SETS). The data includes PRP name and address, a company contact person, the date the notice was issued, and the related CERCLA site name and identification number

  18. Superfund TIO videos. Set B. Basics of administrative law, and prp search process: PRP search, information exchange and access. Part 3. Audio-Visual

    International Nuclear Information System (INIS)

    1990-01-01

    The videotape is divided into two sections. Section 1 identifies the various types of administrative hearings, including quasi-legislative, quasi-judicial, and hybrid types. Section 2 provides an overview of the PRP search process; explains how and when to issue Section 104(e) letters and administrative subpoenas; outlines the enforcement authorities available in cases of non-compliance; and describes the types of information that can be released to PRPs

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

    Science.gov (United States)

    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.

  20. Loss of CDKL5 in Glutamatergic Neurons Disrupts Hippocampal Microcircuitry and Leads to Memory Impairment in Mice.

    Science.gov (United States)

    Tang, Sheng; Wang, I-Ting Judy; Yue, Cuiyong; Takano, Hajime; Terzic, Barbara; Pance, Katarina; Lee, Jun Y; Cui, Yue; Coulter, Douglas A; Zhou, Zhaolan

    2017-08-02

    Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory. SIGNIFICANCE STATEMENT Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although Cdkl5 constitutive knock-out mice have recapitulated key aspects of human symptomatology, the cellular origins of CDKL5 deficiency-related phenotypes are unknown. Here, using conditional knock-out mice, we show that hippocampal-dependent learning and memory deficits in CDKL5 deficiency have origins in glutamatergic neurons of the forebrain and that loss of CDKL5 results in the enhancement of synaptic transmission and disruptions in neural circuit dynamics in a spatially and temporally specific manner. Our findings demonstrate that CDKL5 is an important regulator of synaptic function in glutamatergic neurons and

  1. Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes.

    Science.gov (United States)

    Malhotra, Angad; Pelletier, Matthew H; Yu, Yan; Walsh, William R

    2013-02-01

    The increased concentration of platelets within platelet-rich plasma (PRP) provides a vehicle to deliver supra-physiologic concentrations of growth factors to an injury site, possibly accelerating or otherwise improving connective tissue regeneration. This potential benefit has led to the application of PRP in several applications; however, inconsistent results have limited widespread adoption in bone healing. This review provides a core understanding of the bone healing mechanisms, and corresponds this to the factors present in PRP. In addition, the current state of the art of PRP preparation, the key aspects that may influence its effectiveness, and treatment outcomes as they relate specifically to bone defect healing are presented. Although PRP does have a sound scientific basis, its use for bone healing appears only beneficial when used in combination with osteoconductive scaffolds; however, neither allograft nor autograft appear to be appropriate carriers. Aggressive processing techniques and very high concentrations of PRP may not improve healing outcomes. Moreover, many other variables exist in PRP preparation and use that influence its efficacy; the effect of these variables should be understood when considering PRP use. This review includes the essentials of what has been established, what is currently missing in the literature, and recommendations for future directions.

  2. PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice

    International Nuclear Information System (INIS)

    Bertuchi, Fernanda R.; Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R.; Cerchiaro, Giselle

    2012-01-01

    Highlights: ► PrP C in solution acts as a radical scavenger. ► PrP C reduces hydrogen peroxide toxicity in astrocytes. ► Increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes. ► PrP C prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP C protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP C in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP C decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu 2+ /ascorbate/H 2 O 2 ), which was demonstrated by approximately 70% less DMPO/OH · . In cultured PrP C -knockout astrocytes from mice, the absence of PrP C caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H 2 O 2 treatment. This rapid increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP C in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  3. A comparison between platelet-rich plasma (PRP and hyaluronate acid on the healing of cartilage defects.

    Directory of Open Access Journals (Sweden)

    Ji Liu

    Full Text Available Platelet-rich plasma (PRP has offered great promise for the treatment of cartilage degradation, and has been proved to have positive effects on the restoration of cartilage lesions. But no comparative work has been done between PRP and hyaluronate acid (HA concerning their restoring effect on cartilage defect, especially by means of animal experiments and histologic assessments. The purpose of the study was to compare the therapeutic effects of P-PRP and HA on osteoarthritis in rabbit knees. Thirty rabbits were used to establish the animal models by creating a cartilage defect of 5 mm in diameter on the condyles of the femurs, and were randomly divided into three groups: the P-PRP group, HA group and the control group. Then each group was treated with P-PRP, HA or saline solution, respectively. Six and twelve weeks later the rabbits were sacrificed and the samples were collected. The platelet number, the concentrations of growth factors of P-PRP and whole blood, and the IL-1β concentration in the joint fluid were investigated, and the histological assessment of the cartilage were performed according to Mankin's scoring system. Micro-CT was also used to evaluate the restoration of subchondral bone. The platelet concentration in P-PRP is 6.8 fold of that in the whole blood. The IL-1β level in the P-PRP group was lower than in the HA group (p<0.01 and in the control group (p<0.01. The restoration of the defected cartilage as well as the subchondral bone was better in the P-PRP group than in the HA group or the control group (P<0.05. Our data showed that P-PRP is better than HA in promoting the restoration of the cartilage and alleviating the arthritis caused by cartilage damage.

  4. Splicing Factor Prp8 Interacts With NES(AR) and Regulates Androgen Receptor in Prostate Cancer Cells.

    Science.gov (United States)

    Wang, Dan; Nguyen, Minh M; Masoodi, Khalid Z; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A; Dhir, Rajiv; Wang, Zhou

    2015-12-01

    Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NES(AR)) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NES(AR), we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NES(AR) export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NES(AR) function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NES(AR). Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NES(AR) and regulates AR function in prostate cancer cells.

  5. Ionic mechanisms of action of prion protein fragment PrP(106-126) in rat basal forebrain neurons.

    Science.gov (United States)

    Alier, Kwai; Li, Zongming; Mactavish, David; Westaway, David; Jhamandas, Jack H

    2010-08-01

    Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion-induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K(+)) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106-126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106-126) peptide, but not the "scrambled" sequence of PrP(106-126), evoked a reduction of whole-cell outward currents in a voltage range between -30 and +30 mV. Reduction of whole-cell outward currents was significantly attenuated in Ca(2+)-free external media and also in the presence of iberiotoxin, a blocker of calcium-activated potassium conductance. PrP(106-126) application also evoked a depression of the delayed rectifier (I(K)) and transient outward (I(A)) potassium currents. By using single cell RT-PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K(v)4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106-126) peptide used at nanomolar concentrations, induces a reduction of specific K(+) channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K(+) channels, and PrP-mediated neurotoxicity.

  6. In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.

    Science.gov (United States)

    Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

    2016-01-01

    The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

  7. Exceptions to the PRP Effect? A Comparison of Prepared and Unconditioned Reflexes

    Science.gov (United States)

    Janczyk, Markus; Pfister, Roland; Wallmeier, Gloria; Kunde, Wilfried

    2014-01-01

    Psychological research has documented again and again marked performance decrements whenever humans perform 2 or more tasks at the same time. In fact, the available evidence seems to suggest that any type of behavior is subject to such limitations. The present experiments employed the psychological refractory period (PRP) paradigm to identify a…

  8. No effects of PRP on ultrasonographic tendon structure and neovascularisation in chronic midportion Achilles tendinopathy

    NARCIS (Netherlands)

    de Vos, R. J.; Weir, A.; Tol, J. L.; Verhaar, J. A. N.; Weinans, H.; van Schie, H. T. M.

    2011-01-01

    To assess whether a platelet-rich plasma (PRP) injection leads to an enhanced tendon structure and neovascularisation, measured with ultrasonographic techniques, in chronic midportion Achilles tendinopathy. Double-blind, randomised, placebo-controlled clinical trial. Sports medical department of The

  9. Acetylcholinesterase triggers the aggregation of PrP 106-126

    International Nuclear Information System (INIS)

    Pera, M.; Roman, S.; Ratia, M.; Camps, P.; Munoz-Torrero, D.; Colombo, L.; Manzoni, C.; Salmona, M.; Badia, A.; Clos, M.V.

    2006-01-01

    Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-β-protein (Aβ) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and Aβ aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs

  10. Mice with a targeted disruption of the Fanconi anemia homolog Fanca.

    Science.gov (United States)

    Cheng, N C; van de Vrugt, H J; van der Valk, M A; Oostra, A B; Krimpenfort, P; de Vries, Y; Joenje, H; Berns, A; Arwert, F

    2000-07-22

    Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer predisposition. Bone marrow failure resulting in pancytopenia is the main cause of death of FA patients. Diagnosis of FA is based on their cellular hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic complementation experiments suggest the involvement of at least eight genes in FA. The gene for complementation group A (FANCA) is defective in the majority of FA patients. We show here that mice deficient of FANCA: are viable and have no detectable developmental abnormalities. The hematological parameters showed a slightly decreased platelet count and a slightly increased erythrocyte mean cell volume in mice at young age, but this did not progress to anemia. Consistent with the clinical phenotype of FA patients, both male and female mice showed hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the knock-out mice exhibited spontaneous chromosomal instability and were hyper-responsive to the clastogenic effect of the crosslinker mitomycin C.

  11. On the importance of Task 1 and error performance measures in PRP dual-task studies

    Science.gov (United States)

    Strobach, Tilo; Schütz, Anja; Schubert, Torsten

    2015-01-01

    The psychological refractory period (PRP) paradigm is a dominant research tool in the literature on dual-task performance. In this paradigm a first and second component task (i.e., Task 1 and Task 2) are presented with variable stimulus onset asynchronies (SOAs) and priority to perform Task 1. The main indicator of dual-task impairment in PRP situations is an increasing Task 2-RT with decreasing SOAs. This impairment is typically explained with some task components being processed strictly sequentially in the context of the prominent central bottleneck theory. This assumption could implicitly suggest that processes of Task 1 are unaffected by Task 2 and bottleneck processing, i.e., decreasing SOAs do not increase reaction times (RTs) and error rates of the first task. The aim of the present review is to assess whether PRP dual-task studies included both RT and error data presentations and statistical analyses and whether studies including both data types (i.e., RTs and error rates) show data consistent with this assumption (i.e., decreasing SOAs and unaffected RTs and/or error rates in Task 1). This review demonstrates that, in contrast to RT presentations and analyses, error data is underrepresented in a substantial number of studies. Furthermore, a substantial number of studies with RT and error data showed a statistically significant impairment of Task 1 performance with decreasing SOA. Thus, these studies produced data that is not primarily consistent with the strong assumption that processes of Task 1 are unaffected by Task 2 and bottleneck processing in the context of PRP dual-task situations; this calls for a more careful report and analysis of Task 1 performance in PRP studies and for a more careful consideration of theories proposing additions to the bottleneck assumption, which are sufficiently general to explain Task 1 and Task 2 effects. PMID:25904890

  12. On the importance of Task 1 and error performance measures in PRP dual-task studies.

    Science.gov (United States)

    Strobach, Tilo; Schütz, Anja; Schubert, Torsten

    2015-01-01

    The psychological refractory period (PRP) paradigm is a dominant research tool in the literature on dual-task performance. In this paradigm a first and second component task (i.e., Task 1 and Task 2) are presented with variable stimulus onset asynchronies (SOAs) and priority to perform Task 1. The main indicator of dual-task impairment in PRP situations is an increasing Task 2-RT with decreasing SOAs. This impairment is typically explained with some task components being processed strictly sequentially in the context of the prominent central bottleneck theory. This assumption could implicitly suggest that processes of Task 1 are unaffected by Task 2 and bottleneck processing, i.e., decreasing SOAs do not increase reaction times (RTs) and error rates of the first task. The aim of the present review is to assess whether PRP dual-task studies included both RT and error data presentations and statistical analyses and whether studies including both data types (i.e., RTs and error rates) show data consistent with this assumption (i.e., decreasing SOAs and unaffected RTs and/or error rates in Task 1). This review demonstrates that, in contrast to RT presentations and analyses, error data is underrepresented in a substantial number of studies. Furthermore, a substantial number of studies with RT and error data showed a statistically significant impairment of Task 1 performance with decreasing SOA. Thus, these studies produced data that is not primarily consistent with the strong assumption that processes of Task 1 are unaffected by Task 2 and bottleneck processing in the context of PRP dual-task situations; this calls for a more careful report and analysis of Task 1 performance in PRP studies and for a more careful consideration of theories proposing additions to the bottleneck assumption, which are sufficiently general to explain Task 1 and Task 2 effects.

  13. On the importance of Task 1 and error performance measures in PRP dual-task studies

    Directory of Open Access Journals (Sweden)

    Tilo eStrobach

    2015-04-01

    Full Text Available The Psychological Refractory Period (PRP paradigm is a dominant research tool in the literature on dual-task performance. In this paradigm a first and second component task (i.e., Task 1 and 2 are presented with variable stimulus onset asynchronies (SOAs and priority to perform Task 1. The main indicator of dual-task impairment in PRP situations is an increasing Task 2-RT with decreasing SOAs. This impairment is typically explained with some task components being processed strictly sequentially in the context of the prominent central bottleneck theory. This assumption could implicitly suggest that processes of Task 1 are unaffected by Task 2 and bottleneck processing, i.e. decreasing SOAs do not increase RTs and error rates of the first task. The aim of the present review is to assess whether PRP dual-task studies included both RT and error data presentations and statistical analyses and whether studies including both data types (i.e., RTs and error rates show data consistent with this assumption (i.e., decreasing SOAs and unaffected RTs and/ or error rates in Task 1. This review demonstrates that, in contrast to RT presentations and analyses, error data is underrepresented in a substantial number of studies. Furthermore, a substantial number of studies with RT and error data showed a statistically significant impairment of Task 1 performance with decreasing SOA. Thus, these studies produced data that is not primarily consistent with the strong assumption that processes of Task 1 are unaffected by Task 2 and bottleneck processing in the context of PRP dual-task situations; this calls for a more careful report and analysis of Task 1 performance in PRP studies and for a more careful consideration of theories proposing additions to the bottleneck assumption, which are sufficiently general to explain Task 1 and Task 2 effects.

  14. Comparison of the effect of activated or non-activated PRP in various concentrations on osteoblast and fibroblast cell line proliferation.

    Science.gov (United States)

    Vahabi, Surena; Yadegari, Zahra; Mohammad-Rahimi, Hossein

    2017-09-01

    Platelet-rich plasma (PRP) contains growth factors which positively affect cell proliferation, cell differentiation, chemotaxis and intracellular matrix synthesis. All these processes are involved in wound healing and tissue regeneration; thus, PRP as a source of growth factors can be used in periodontal regenerative therapies. The purpose of the present study was to assess the effect of various concentrations of activated and non-activated PRP on proliferation of osteoblasts and fibroblasts in vitro. PRP was obtained from three healthy volunteers. 75, 50, 25, and 10% concentrations of f PRP were prepared by dilution in Dulbecco's modified Eagle's medium. In activated PRP groups, PRP concentrations were activated by adding calcium gluconate. Human gingival fibroblast (HGF) cell line and MG-63 (osteosarcoma) human osteoblast-like cell line were used in the study. The MTT proliferation assay was used to assess the effect of different types of PRP concentrates on proliferation of HGF and MG-63 cells, in 24, 48 and 72 h. After 24, 48, and 72 h, the proliferation rate of both cell lines was higher in the positive control group, except in 72 h in HGF cell lines, that 10% non-activated PRP group and 10 and 25% activated PRP groups has higher proliferation rate than the positive control group, which it was not significant. Proliferation rate in cells with 10% activated PRP was highest among samples containing PRP. The current study failed to show the significant effect of activated or non-activated PRP on proliferation of HGFs or MG-63 osteoblast-like cells. However, our results showed that activated PRP had a greater effect than non-activated PRP.

  15. Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro.

    Science.gov (United States)

    Zheng, Canbin; Zhu, Qingtang; Liu, Xiaolin; Huang, Xijun; He, Caifeng; Jiang, Li; Quan, Daping; Zhou, Xiang; Zhu, Zhaowei

    2016-05-01

    Platelet-rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5-20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose-dependently. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

    Science.gov (United States)

    Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

    2015-06-01

    The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

  17. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

    Science.gov (United States)

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio

    2012-01-01

    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

  18. Interaction between Shadoo and PrP Affects the PrP-Folding Pathway.

    Science.gov (United States)

    Ciric, Danica; Richard, Charles-Adrien; Moudjou, Mohammed; Chapuis, Jérôme; Sibille, Pierre; Daude, Nathalie; Westaway, David; Adrover, Miguel; Béringue, Vincent; Martin, Davy; Rezaei, Human

    2015-06-01

    Prion diseases are characterized by conformational changes of a cellular prion protein (PrP(C)) into a β-sheet-enriched and aggregated conformer (PrP(Sc)). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP with a dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrP(Sc) conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by (i) acting as a holdase and (ii) interfering with the dominant-negative inhibitor effect of the C1 fragment. Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterization of Sho-PrP indicates that Sho acts as an ATP-independent holdase. Copyright © 2015, American Society for Microbiology. All Rights

  19. L-citrulline protects from kidney damage in type 1 diabetic mice.

    Directory of Open Access Journals (Sweden)

    Maritza J Romero

    2013-12-01

    Full Text Available Rationale. Diabetic nephropathy is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of L-arginine (L-arg, the substrate for endothelial nitric oxide synthase (eNOS, failed to improve vascular function. L-citrulline (L-cit supplementation not only increases L-arg synthesis, but also inhibits cytosolic arginase I (Arg I, a competitor of eNOS for the use of L-arg, in the vasculature. Aims. To investigate whether L-cit treatment reduces diabetic nephropathy in streptozotocin (STZ-induced type 1 diabetes in mice and rats and to study its effects on arginase II (ArgII function, the main renal isoform. Methods. STZ-C57BL6 mice received L-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and L-cit-treated STZ-rats were evaluated. Results. L-cit exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, L-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 wks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater BUN levels, hypertrophy, and dilated tubules than diabetic wild type mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic wild type animals. L-cit also restored NO/ROS balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, L-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1beta and IL-12(p70 generation in the human proximal tubular cells. Conclusions. L-cit supplementation established an anti-inflammatory profile and significantly preserved the nephron function during type 1

  20. Tendinopathies and platelet-rich plasma (PRP: from pre-clinical experiments to therapeutic use

    Directory of Open Access Journals (Sweden)

    Kaux JF

    2015-05-01

    Full Text Available Objectives: The restorative properties of platelets, through the local release of growth factors, are used in various medical areas. This article reviews fundamental and clinical research relating to platelet-rich plasma applied to tendinous lesions. Materials and method: Articles in French and English, published between 1 January 2012 and 31 December 2014. dealing with PRP and tendons were searched for using the Medline and Scopus data bases. Results: Forty-seven articles were identified which addressed pre-clinical and clinical studies: 27 relating to in vitro and in vivo animal studies and 20 relating to human studies. Of these, five addressed lateral epicondylitis, two addressed rotator cuff tendinopathies, ten dealt with patellar tendinopathies and three looked at Achilles tendinopathies. Conclusions: The majority of pre-clinical studies show that PRP stimulates the tendon's healing process. However, clinical series remain more controversial and level 1, controlled, randomised studies are still needed.

  1. Is PRP useful in alveolar cleft reconstruction? Platelet-rich plasma in secondary alveoloplasty.

    Science.gov (United States)

    Luaces-Rey, Ramon; Arenaz-Búa, Jorge; Lopez-Cedrún-Cembranos, José-Luis; Herrero-Patiño, Susana; Sironvalle-Soliva, Sheyla; Iglesias-Candal, Emma; Pombo-Castro, María

    2010-07-01

    Cleft lip and palate is a congenital facial malformation with an established treatment protocol. Mixed dentition period is the best moment for correct maxillary bone defect with an alveoloplasty. The aim of this surgical procedure is to facilitate dental eruption, re-establish maxillary arch, close any oro-nasal communication, give support to nasal ala, and in some cases allow dental rehabilitation with osteointegrated implants. Twenty cleft patients who underwent secondary alveoloplasty were included. In 10 of them autogenous bone graft were used and in other 10 autogenous bone and platelet-rich plasma (PRP) obtained from autogenous blood. Bone formation was compared by digital orthopantomography made on immediate post-operatory and 3 and 6 months after the surgery. No significant differences were found between both therapeutic groups on bone regeneration. We do not find justified the use of PRP for alveoloplasty in cleft patients' treatment protocol.

  2. Platelet-rich plasma (PRP for the treatment of vulvar lichen sclerosus in a premenopausal woman: A case report

    Directory of Open Access Journals (Sweden)

    D. Franic

    2018-04-01

    Full Text Available The use of platelet-rich plasma (PRP for the treatment of lichen sclerosus (LS in a 38-year-old premenopausal woman is reported. The diagnosis was confirmed histologically and the symptoms documented using the ICIQ Vaginal Symptoms Questionnaire (ICIQ-VS and the Female Sexual Function Index (FSFI questionnaire. PRP was prepared from autologous blood using the Regen Cellular Matrix Kit. PRP was administered twice over two months. Histology at follow-up one month after the second administration showed the epidermis was nearly normal and upper dermal cellularity had been restored. The patient was symptom-free and both her ICIQ-VS and her FSFI scores had improved significantly. PRP is a potential new treatment option for LS which needs further assessment in randomized controlled trials. Keywords: Platelet-rich plasma, Vulvar lichen sclerosus, Premenopause, Treatment

  3. Hepatic farnesoid X-receptor isoforms α2 and α4 differentially modulate bile salt and lipoprotein metabolism in mice.

    Directory of Open Access Journals (Sweden)

    Marije Boesjes

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

  4. Alterations in cell growth and signaling in ErbB3 binding protein-1 (Ebp1 deficient mice

    Directory of Open Access Journals (Sweden)

    Lee Myounghee

    2008-12-01

    Full Text Available Abstract Background The ErbB3 binding protein-1 (Ebp1 belongs to a family of DNA/RNA binding proteins implicated in cell growth, apoptosis and differentiation. However, the physiological role of Ebp1 in the whole organism is not known. Therefore, we generated Ebp1-deficient mice carrying a gene trap insertion in intron 2 of the Ebp1 (pa2g4 gene. Results Ebp1-/- mice were on average 30% smaller than wild type and heterozygous sex matched littermates. Growth retardation was apparent from Day 10 until Day 30. IGF-1 production and IGBP-3 and 4 protein levels were reduced in both embryo fibroblasts and adult knock-out mice. The proliferation of fibroblasts derived from Day 12.5 knock out embryos was also decreased as compared to that of wild type cells. Microarray expression analysis revealed changes in genes important in cell growth including members of the MAPK signal transduction pathway. In addition, the expression or activation of proliferation related genes such as AKT and the androgen receptor, previously demonstrated to be affected by Ebp1 expression in vitro, was altered in adult tissues. Conclusion These results indicate that Ebp1 can affect growth in an animal model, but that the expression of proliferation related genes is cell and context specific. The Ebp1-/- mouse line represents a new in vivo model to investigate Ebp1 function in the whole organism.

  5. Using PRP and human amniotic fluid combination for osteogenesis in rabbit socket preservation

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    Amir Hossein Moradi

    2015-01-01

    Full Text Available Introduction: Platelet-rich plasma (PRP is used as an adjunct treatment during periodontal grafting surgery because of its capability of enhancing healing process. Amniotic fluid is a rich source of growth factors and hyaluronic acid (HA and a good point to study its properties of wound healing and bone formation. The aim of this study was to evaluate the osteogenic properties of a combination of amniotic fluid and PRP in rabbit′s dental socket preservation. Materials and Methods: The study population consisted of 24 healthy male laboratory rabbits (average weight 3,125 ± 185 gr that were randomly allocated into four groups. PRP for the first group, human amniotic fluid (HAF for the second group, a combination of PRP and HAF (PRHA for the third group was used. In the fourth (control group, no biomaterial was used. In each group, half of the rabbits were sacrificed at 4 weeks following surgery and the rest were sacrificed after 8 weeks. Histological analysis of biopsies of the sockets was performed using hematoxylin and eosin (H&E staining. Data were analyzed using Statistical Package for the Social Sciences (SPSS software (version 16 and P-value <0.05 was considered significance. Results: All three experimental groups showed positive effect on bone formation in terms of area of trabecular bone and number of osteocytes and also vessel formation. Socket preservation using HAF and PRHA showed the highest impact on bone formation. Socket preservation using HAF also had the highest impact on vessel formation. Conclusion: PRHA and HAF appear to be useful for enhancing bone formation. Since there was no difference between HAF and PRHA, it seems beneficial to use HAF due to its simplicity of application.

  6. Oxidation of Helix-3 methionines precedes the formation of PK resistant PrP.

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    Tamar Canello

    2010-07-01

    Full Text Available While elucidating the peculiar epitope of the alpha-PrP mAb IPC2, we found that PrPSc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the alpha-fold, suggesting oxidation may facilitate the conversion of PrPC into PrPSc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrPC, but could not recognize most PrPSc bands unless they were vigorously reduced. Next, we showed that the alpha-Met pAbs did not recognize newly formed PrPSc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrPC to PrPSc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

  7. Efectividad del PRP en el tratamiento de la artrosis de rodilla: Estudio de casos

    OpenAIRE

    Fernández Valverde, Noelia; Fidalgo González, Verónica

    2016-01-01

    La artrosis es una enfermedad degenerativa de elevada prevalencia para la cual, en la actualidad, no hay tratamiento curativo. Una de las localizaciones más frecuentes es la rodilla. La utilización de infiltraciones de plasma rico en plaquetas constituye una alternativa en el tratamiento de esta patología. Objetivo: demostrar la efectividad y la seguridad de las infiltraciones con PRP en el tratamiento de pacientes con artrosis de rodilla. Resultado: este trabajo muestra una...

  8. Platelet-rich plasma (PRP) in dental and oral surgery: from the wound healing to bone regeneration

    Science.gov (United States)

    2013-01-01

    Platelet-rich plasma (PRP) is a new approach to tissue regeneration and it is becoming a valuable adjunct to promote healing in many procedures in dental and oral surgery, especially in aging patients. PRP derives from the centrifugation of the patient's own blood and it contains growth factors that influence wound healing, thereby playing an important role in tissue repairing mechanisms. The use of PRP in surgical practice could have beneficial outcomes, reducing bleeding and enhancing soft tissue healing and bone regeneration. Studies conducted on humans have yielded promising results regarding the application of PRP to many dental and oral surgical procedures (i.e. tooth extractions, periodontal surgery, implant surgery). The use of PRP has also been proposed in the management of bisphosphonate-related osteonecrosis of the jaw (BRONJ) with the aim of enhancing wound healing and bone maturation. The aims of this narrative review are: i) to describe the different uses of PRP in dental surgery (tooth extractions and periodontal surgery) and oral surgery (soft tissues and bone tissue surgery, implant surgery and BRONJ surgery); and ii) to discuss its efficacy, efficiency and risk/benefit ratio. This review suggests that the use of PRP in the alveolar socket after tooth extractions is certainly capable of improving soft tissue healing and positively influencing bone regeneration but the latter effect seems to decrease a few days after the extraction. PRP has produced better results in periodontal therapy in association with other materials than when it is used alone. Promising results have also been obtained in implant surgery, when PRP was used in isolation as a coating material. The combination of necrotic bone curettage and PRP application seem to be encouraging for the treatment of refractory BRONJ, as it has proven successful outcomes with minimal invasivity. Since PRP is free from potential risks for patients, not difficult to obtain and use, it can be employed

  9. Platelet-rich plasma (PRP) in dental and oral surgery: from the wound healing to bone regeneration.

    Science.gov (United States)

    Albanese, Antonino; Licata, Maria E; Polizzi, Bianca; Campisi, Giuseppina

    2013-06-13

    Platelet-rich plasma (PRP) is a new approach to tissue regeneration and it is becoming a valuable adjunct to promote healing in many procedures in dental and oral surgery, especially in aging patients. PRP derives from the centrifugation of the patient's own blood and it contains growth factors that influence wound healing, thereby playing an important role in tissue repairing mechanisms. The use of PRP in surgical practice could have beneficial outcomes, reducing bleeding and enhancing soft tissue healing and bone regeneration. Studies conducted on humans have yielded promising results regarding the application of PRP to many dental and oral surgical procedures (i.e. tooth extractions, periodontal surgery, implant surgery). The use of PRP has also been proposed in the management of bisphosphonate-related osteonecrosis of the jaw (BRONJ) with the aim of enhancing wound healing and bone maturation. The aims of this narrative review are: i) to describe the different uses of PRP in dental surgery (tooth extractions and periodontal surgery) and oral surgery (soft tissues and bone tissue surgery, implant surgery and BRONJ surgery); and ii) to discuss its efficacy, efficiency and risk/benefit ratio. This review suggests that the use of PRP in the alveolar socket after tooth extractions is certainly capable of improving soft tissue healing and positively influencing bone regeneration but the latter effect seems to decrease a few days after the extraction. PRP has produced better results in periodontal therapy in association with other materials than when it is used alone. Promising results have also been obtained in implant surgery, when PRP was used in isolation as a coating material. The combination of necrotic bone curettage and PRP application seem to be encouraging for the treatment of refractory BRONJ, as it has proven successful outcomes with minimal invasivity. Since PRP is free from potential risks for patients, not difficult to obtain and use, it can be employed

  10. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage.

    Science.gov (United States)

    Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

    2017-03-01

    Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. The role of PRP and adipose tissue-derived keratinocytes on burn wound healing in diabetic rats.

    Science.gov (United States)

    Hosseini Mansoub, Navid; Gürdal, Mehmet; Karadadaş, Elif; Kabadayi, Hilal; Vatansever, Seda; Ercan, Gulinnaz

    2018-01-01

    Introduction: Diabetic burn wounds and ulcers are significant complications of diabetic patients. The aim of this study is to investigate the use of platelet rich-plasma (PRP) and/or keratinocyte-like cells (KLCs) in diabetic thermal wound rat model and to evaluate EGF, FGF-2, TGF-β1, COL1α2, MCP-1 and VEGF-α as wound healing markers at gene expression level. Method: In this study, we used adipose tissue as the source of mesenchymal stem cells (MSCs) and differentiated MSCs into KLCs. KLCs were characterized and transferred to the burn areas on the dorsum of streptozotocine (STZ)-induced diabetic rats. We prepared PRP from rat blood and evaluated its effect alone or in combination with KLCs. On 3 rd , 7 th , 10 th and 14 th days after treatment, wound areas were measured and biopsy samples were excised from the wound areas of the KLCs and/or PRP-treated and untreated diabetic rats to analyze gene expression levels of wound healing markers by qPCR. Results: We observed that, wound contraction started earlier in the PRP and/or KLCs-treated groups in comparison to the control group. However, PRP and KLCs when applied in combination showed additive affect in wound healing. In all groups treated with KLCs and/or PRP, the gene expression levels of evaluated growth factors and COL1α2 increased, while MCP-1 levels decreased when compared to the untreated diabetic rats. In addition, the most prominent difference in qPCR results belongs to combined PRP and KLCs-treated group. Conclusion: We demonstrated that applying PRP and KLCs in combination has a greater potential for treatment of diabetic burn wounds.

  12. Platelet-rich plasma (PRP for acute muscle injury: a systematic review.

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    Mohamad Shariff A Hamid

    Full Text Available INTRODUCTION: Acute muscle injury is one of the commonest injuries that often result in loss of training and competition time. The best management for muscle injury has not been identified. Sports medicine practitioners used several approaches in attempt to accelerate time to recovery from muscle injury. More recently growing interest focussed on autologous blood product injection. METHODS: A literature search was conducted systematically using OvidMEDLINE, PubMed, EMBASE, SPORTDiscus and CINAHL databases to retrieve articles published until December 2012. Controlled trials and controlled laboratory studies comparing different strategies to promote early recovery of muscle injury were included. The methodological quality of studies was assessed. RESULTS: There are limited studies on the effects of PRP therapy for muscle injury. Three in vivo laboratory studies and one pilot human study were reviewed. The laboratory studies reported histological evidence on significant acceleration of muscle healing in animals treated with autologous conditioned serum (ACS, platelet-rich plasma (PRP and platelet rich fibrin matrix (PRFM. A pilot human study found athletes treated with repeated ACS injection recovers significantly faster than retrospective controls. CONCLUSION: Several in vivo laboratory studies suggest beneficial effects of ACS, PRP and PRFM in accelerating muscle recovery. Evidence to suggest similar effects on humans is however limited, as valuable information from robust human controlled trials is still not available at this moment. Hence, more studies of satisfactory methodological quality with platelet-rich plasma interventions on muscle injury are justified.

  13. Comparison between PRP, PRGF and PRF: lights and shadows in three similar but different protocols.

    Science.gov (United States)

    Giannini, S; Cielo, A; Bonanome, L; Rastelli, C; Derla, C; Corpaci, F; Falisi, G

    2015-01-01

    The main goal of the modern surgery is to get a low invasiveness and a high rate of clinical healing: in the last years, it has been introduced the concept of a "regenerative surgery", and many techniques has been widely described in the literature. The most used are PRP, PRGF and PRF techniques. Aim of this research is to compare the three protocol of PRP, PRF and PRGF in their essential features, so to suggest to the practitioners the best blood product to use in the regenerative surgery. Among the advantages that shows the PRF, compared to PRP and PRGF, we can cite a greater simplicity of production for the absence of manipulation that leads to a reduced possibility of alteration of the protocol due to an error of the operator. The special texture of the PRF and its biological features shows clearly an interesting surgical versatility and all the characteristics that can support a faster tissues regeneration and high-quality clinical outcomes.

  14. Autologous neural progenitor cell transplantation into newborn mice modeling for E200K genetic prion disease delays disease progression.

    Science.gov (United States)

    Frid, Kati; Binyamin, Orli; Fainstein, Nina; Keller, Guy; Ben-Hur, Tamir; Gabizon, Ruth

    2018-05-01

    TgMHu2ME199K mice, a transgenic line mimicking genetic prion disease, are born healthy and gradually deteriorate to a terminal neurological condition concomitant with the accumulation of disease-related PrP. To investigate whether transplantation of neural progenitor cells (NPCs) to these mice can delay disease aggravation, we first tested the properties of mutant PrP in homogenates and enriched NPCs from TgMHu2ME199K embryos, as compared to PrP in sick TgMHu2ME199K brains. Next, we tested the clinical effect of NPCs transplantation into newborn TgMHu2ME199K mice. We show that mutant PrP does not convert into a disease-related isoform while in progenitor cells. Most important, transplantation of both wild type and transgenic NPCs significantly delayed the progression of spontaneous prion disease in TgMHu2ME199K mice. While the strong clinical effect was not accompanied by a reduced accumulation of disease-related PrP, treated mouse brains presented a significant reduction in amyloid glycosaminoglycans and preservation of neurogenesis levels, indicating a strong neuroprotective effect. These results may encourage the investigation of new pathways for treatment in these terrible diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. In Vitro Studies on the Degradability, Bioactivity, and Cell Differentiation of PRP/AZ31B Mg Alloys Composite Scaffold

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    Jian Zou

    2017-01-01

    Full Text Available In recent years, more and more methods have been developed to improve the bioactivity of the biodegradable materials in bone tissue regeneration. In present study, we used rat mesenchymal stem cells (rMSCs to evaluate the outcomes of Mg alloys (AZ31B, Magnesium, and Aluminum and Platelet-rich plasma (PRP/Mg alloys on rMSCs biocompatibility and osteogenic differentiation. Water absorption experiments indicated that both bare AZ31B and PRP/AZ31B were capable of absorbing large amounts of water. But the water absorption ratio for PRP/AZ31B was significantly higher than that for bare AZ31B. The degradability experiments implied that both samples degraded at same speed. rMSCs on the surface of AZ31B distributed more and better than those on the AZ31B scaffold. In ALP activity experiment, the activity of rMSCs on the PRP/AZ31B was markedly higher than that on the AZ31B scaffolds on the 7th day and 14th day. qRT-PCR also showed that OPN and OCN were expressed in both samples. OPN and OCN expression in PRP/AZ31B sample were higher than those in bare AZ31B samples. In summary, the in vitro study implied that AZ31B combined with PRP could remarkably improve cell seeding, attachment, proliferation, and differentiation.

  16. Prion protein is a key determinant of alcohol sensitivity through the modulation of N-methyl-D-aspartate receptor (NMDAR activity.

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    Agnès Petit-Paitel

    Full Text Available The prion protein (PrP is absolutely required for the development of prion diseases; nevertheless, its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches in transgenic mouse models, we provide strong evidence for a crucial role of PrP in alcohol sensitivity. Indeed, PrP knock out (PrP(-/- mice presented a greater sensitivity to the sedative effects of EtOH compared to wild-type (wt control mice. Conversely, compared to wt mice, those over-expressing mouse, human or hamster PrP genes presented a relative insensitivity to ethanol-induced sedation. An acute tolerance (i.e. reversion to ethanol inhibition of N-methyl-D-aspartate (NMDA receptor-mediated excitatory post-synaptic potentials in hippocampal slices developed slower in PrP(-/- mice than in wt mice. We show that PrP is required to induce acute tolerance to ethanol by activating a Src-protein tyrosine kinase-dependent intracellular signaling pathway. In an attempt to decipher the molecular mechanisms underlying PrP-dependent ethanol effect, we looked for changes in lipid raft features in hippocampus of ethanol-treated wt mice compared to PrP(-/- mice. Ethanol induced rapid and transient changes of buoyancy of lipid raft-associated proteins in hippocampus of wt but not PrP(-/- mice suggesting a possible mechanistic link for PrP-dependent signal transduction. Together, our results reveal a hitherto unknown physiological role of PrP on the regulation of NMDAR activity and highlight its crucial role in synaptic functions.

  17. Tratamiento de quemaduras mediante plasma rico en plaquetas (PRP: parte I

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    G. Rossani

    2014-06-01

    Full Text Available El objetivo del presente trabajo es determinar la eficacia clínica del plasma rico en plaquetas (PRP en las quemaduras de segundo grado. Estudiamos el tiempo requerido en la reepitelización del tejido dañado, la estancia hospitalaria asociada a la curación de las lesiones y la satisfacción del paciente. Realizamos un estudio prospectivo, observacional y longitudinal, en una muestra de 115 pacientes con quemaduras de segundo grado según la clasificación de Converse-Smith. Las lesiones fueron de menos de 48 horas de evolución, en diferentes zonas de cara y cuerpo. A todos los pacientes se les aplicó de forma ambulatoria PRP por goteo, completándose el tratamiento con la aplicación de gasas parafinadas. El estudio se realizó entre marzo de 2011 y agosto de 2013. Las quemaduras que evolucionaron mejor y de forma más rápida fueron las de cara, seguidas por las de abdomen y, por último, las de extremidades inferiores. En todas, el tiempo de epitelización fue un 30 % inferior que en quemaduras de similar extensión, profundidad y localización, en pacientes anteriormente tratados sin PRP. Los pacientes fueron atendidos ambulatoriamente cuando las lesiones lo permitieron, y si presentaban lesiones más extensas fueron hospitalizados. El tiempo de internamiento en estos casos se redujo como promedio 18 días con respecto al grupo no tratado con PRP. El tiempo de reepitelización, estancia hospitalaria y la satisfacción de los pacientes, alcanzaron significación estadística p< 0,05. En conclusión, creemos que el uso de PRP acorta el tiempo de recuperación en quemaduras de segundo grado, reduce el tiempo de hospitalización y conlleva un alto grado de satisfacción de los pacientes por los resultados obtenidos.

  18. PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions.

    Science.gov (United States)

    Kraus, Allison; Raymond, Gregory J; Race, Brent; Campbell, Katrina J; Hughson, Andrew G; Anson, Kelsie J; Raymond, Lynne D; Caughey, Byron

    2017-11-01

    Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro , only some are transmissible and pathogenic in vivo To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids. IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  20. Split face comparative study of microneedling with PRP versus microneedling with vitamin C in treating atrophic post acne scars

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    Simran Chawla

    2014-01-01

    Full Text Available Introduction: Acne scars are largely preventable complications of acne. 95% of the scars occur over the face thus impacting the quality of life. Correction of scars is the priority for acne patients. Materials and Methods: Thirty patients with post acne atrophic facial scars attending the OPD during the period from April to October 2013 were offered four sittings of microneedling with PRP on one side and microneedling with vitamin C on other side of the face at an interval of 1 month. Results: Twenty-seven out of the total 30 patients completed the treatment schedule. Two patients were lost to follow up and one dropped out of the study due to severe PIH. Mean age of the patients was 27.5 years. Out of 30 patients, 23 achieved reduction in scarring by one or two grades. Excellent response was seen in five (18.5% patients with platelet-rich plasma (PRP as compared to two (7% patients who received treatment with vitamin C according to physician′s assessment. As far as up gradation by 1 score is considered, i.e., good response, it was similar in both cases. Vitamin C did not prove to be as efficacious as PRP since 10 (37% patients had poor response in vitamin C-treated area compared to only 6 (22.2% patients who underwent PRP therapy, but vitamin C proved to be efficacious in dealing with post inflammatory hyper-pigmentation secondary to acne. Patients were more satisfied with PRP as compared to vitamin C. The results were evaluated and statistical analysis was done using SPSS 16.0.2. Conclusions: Overall results were better with microneedling and PRP. Vitamin C combined with microneedling also showed improvement with respect to firmness and smoothness of skin; as well as post inflammatory hyper-pigmentation. Microneedling combined with PRP proved to be good in treating boxcar and rolling scars but had limited efficacy in dealing with ice pick scars.

  1. Expression of inactive glutathione peroxidase 4 leads to embryonic lethality, and inactivation of the Alox15 gene does not rescue such knock-in mice.

    Science.gov (United States)

    Brütsch, Simone Hanna; Wang, Chi Chiu; Li, Lu; Stender, Hannelore; Neziroglu, Nilgün; Richter, Constanze; Kuhn, Hartmut; Borchert, Astrid

    2015-02-01

    Glutathione peroxidases (Gpx) and lipoxygenases (Alox) are functional counterplayers in the metabolism of hydroperoxy lipids that regulate cellular redox homeostasis. Gpx4 is a moonlighting protein that has been implicated not only as an enzyme in anti-oxidative defense, gene expression regulation, and programmed cell death, but also as a structural protein in spermatogenesis. Homozygous Gpx4 knock-out mice are not viable, but molecular reasons for intrauterine lethality are not completely understood. This study was aimed at investigating whether the lack of catalytic activity or the impaired function as structural protein is the dominant reason for embryonic lethality. We further explored whether the pro-oxidative enzyme mouse 12/15 lipoxygenase (Alox15) plays a major role in embryonic lethality of Gpx4-deficient mice. To achieve these goals, we first created knock-in mice, which express a catalytically inactive Gpx4 mutant (Sec46Ala). As homozygous Gpx4-knock-out mice Sec46Ala-Gpx4(+/+) knock-in animals are not viable but undergo intrauterine resorption between embryonic day 6 and 7 (E6-7). In contrast, heterozygous knock-in mice (Sec46Ala-Gpx4(-/+)) are viable, fertile and do not show major phenotypic alterations. Interestingly, homozygous Alox15 deficiency did not rescue the U46A-Gpx4(+/+) mice from embryonic lethality. In fact, when heterozygous U46A-Gpx4(-/+) mice were stepwise crossed into an Alox15-deficent background, no viable U46A-Gpx4(+/+)+Alox15(-/-) individuals were obtained. However, we were able to identify U46A-Gpx4(+/+)+Alox15(-/-) embryos in the state of resorption around E7. These data suggest that the lack of catalytic activity is the major reason for the embryonic lethality of Gpx4(-/-) mice and that systemic inactivation of the Alox15 gene does not rescue homozygous knock-in mice expressing catalytically silent Gpx4.

  2. Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

    Science.gov (United States)

    Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-01-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

  3. Transcription factor YY1 can control AID-mediated mutagenesis in mice.

    Science.gov (United States)

    Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

    2018-02-01

    Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Tratamiento de quemaduras mediante plasma rico en plaquetas (PRP): parte I

    OpenAIRE

    G. Rossani; I. Hernández; J.M. Alcolea; R. Castro-Sierra; W. Pérez-Soto; M.A. Trelles

    2014-01-01

    El objetivo del presente trabajo es determinar la eficacia clínica del plasma rico en plaquetas (PRP) en las quemaduras de segundo grado. Estudiamos el tiempo requerido en la reepitelización del tejido dañado, la estancia hospitalaria asociada a la curación de las lesiones y la satisfacción del paciente. Realizamos un estudio prospectivo, observacional y longitudinal, en una muestra de 115 pacientes con quemaduras de segundo grado según la clasificación de Converse-Smith. Las lesiones fueron ...

  5. Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

    Directory of Open Access Journals (Sweden)

    Manal Alkan

    2015-01-01

    Full Text Available Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD mouse model. To this end, we used mice (inactivated knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response.

  6. Platelet-Rich Plasma (PRP) Rinses for the Treatment of Non-Responding Oral Lichen Planus: A Case Report.

    Science.gov (United States)

    Merigo, Elisabetta; Oppici, Aldo; Parlatore, Anna; Cella, Luigi; Clini, Fabio; Fontana, Matteo; Fornaini, Carlo

    2018-02-06

    Platelet-rich plasma (PRP) has been proposed for different applications in the medical field and in maxillofacial surgery thanks to its many growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and keratinocyte growth factor (KGF). Oral lichen planus (OLP) is a disease that affects the oral mucosa in a chronic way. This disease frequently worsens the quality of life of patients, particularly when clinical manifestations are of the erythematous or erosive/ulcerative type. The properties of PRP that are supported by scientific literature in both oral medicine and other medical fields have suggested the introduction of PRP in clinical practice for the medical treatment of different soft tissues diseases, such as when OLP patients do not respond to conventional therapies, or when conventional treatments have some contraindications or side effects. The aim of this work is to describe the use of PRP used as an oral rinse for the treatment of a patient diagnosed as affected by OLP at the Dentistry, Special Needs and Maxillo-Facial Surgery Unit of the Hospital of Piacenza. PRP protocol was started after the failure of conventional therapies based on the use of topical and systemic corticosteroids, hydroxychloroquine, and low-level laser therapy applications.

  7. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

    Energy Technology Data Exchange (ETDEWEB)

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-09-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  8. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

    Science.gov (United States)

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-01-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  9. Platelet-Rich Plasma (PRP Rinses for the Treatment of Non-Responding Oral Lichen Planus: A Case Report

    Directory of Open Access Journals (Sweden)

    Elisabetta Merigo

    2018-02-01

    Full Text Available Platelet-rich plasma (PRP has been proposed for different applications in the medical field and in maxillofacial surgery thanks to its many growth factors, such as epidermal growth factor (EGF, fibroblast growth factor (FGF, and keratinocyte growth factor (KGF. Oral lichen planus (OLP is a disease that affects the oral mucosa in a chronic way. This disease frequently worsens the quality of life of patients, particularly when clinical manifestations are of the erythematous or erosive/ulcerative type. The properties of PRP that are supported by scientific literature in both oral medicine and other medical fields have suggested the introduction of PRP in clinical practice for the medical treatment of different soft tissues diseases, such as when OLP patients do not respond to conventional therapies, or when conventional treatments have some contraindications or side effects. The aim of this work is to describe the use of PRP used as an oral rinse for the treatment of a patient diagnosed as affected by OLP at the Dentistry, Special Needs and Maxillo-Facial Surgery Unit of the Hospital of Piacenza. PRP protocol was started after the failure of conventional therapies based on the use of topical and systemic corticosteroids, hydroxychloroquine, and low-level laser therapy applications.

  10. Sex-dependent novelty response in neurexin-1α mutant mice.

    Directory of Open Access Journals (Sweden)

    Marijke C Laarakker

    Full Text Available Neurexin-1 alpha (NRXN1α belongs to the family of cell adhesion molecules (CAMs, which are involved in the formation of neuronal networks and synapses. NRXN1α gene mutations have been identified in neuropsychiatric diseases including Schizophrenia (SCZ and Autism Spectrum Disorder (ASD. In order to get a better understanding of the pleiotropic behavioral manifestations caused by NRXN1α gene mutations, we performed a behavioral study of Nrxn1α heterozygous knock-out (+/- mice and observed increased responsiveness to novelty and accelerated habituation to novel environments compared to wild type (+/+ litter-mates. However, this effect was mainly observed in male mice, strongly suggesting that gender-specific mechanisms play an important role in Nrxn1α-induced phenotypes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hong-Tao; Liu, Hua; Gao, Xiao-Shu [Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032 (China); Zhang, Hongxia, E-mail: hxzhang@sippe.ac.cn [Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032 (China)

    2009-05-08

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

  12. Polarization phenomena in knock-out reactions and the structure of the deuteron

    International Nuclear Information System (INIS)

    Kolybasov, V.M.

    1996-01-01

    Basic picture is given for polarization phenomena in quasi-free processes. It can be used as simple and universal starting point for polarization investigations. The generalization of Treiman-Yang test serves for the identification of the reaction mechanism. The expressions for above-mentioned characteristics show the way to obtain new information on D-wave deuteron function and thereby to refine tensor terms of N N-potential [ru

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

    Science.gov (United States)

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

    2014-04-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  15. Knock-Out and Transgenic Strategies to Improve Neural Transplantation Therapy for Parkinson's Disease

    National Research Council Canada - National Science Library

    Isacson, Ole

    2001-01-01

    .... To enhance axonal growth leading to optimal functional recovery by neuronal transplants, we employed transgenic bcl-2 overexpressing donor cells and similar molecules influencing the growth of axons...

  16. Knock-out and Transgenic Strategies to Improve Neural Transplantation Therapy for Parkinson's Disease

    National Research Council Canada - National Science Library

    Isacson, Ole

    2000-01-01

    .... In our second objective of enhancing axonal growth leading to optimal functional recovery by neuronal transplants, we employed transgenic bcl-2 overexpressing donor cells and similar molecules...

  17. Characterisation of enterocolitis in the piroxicam-accelerated interleukin-10 knock out mouse

    DEFF Research Database (Denmark)

    Holgersen, Kristine; Kvist, Peter Helding; Markholst, Helle

    2014-01-01

    Background: In inflammatory bowel disease a defective mucosal barrier, a dysregulated immune response and an excessive reactivity against the gut microbiota are assumed to cause a breakdown of the intestinal homeostasis and lead to chronic inflammation. Piroxicam treatment is a method for inducti...

  18. Role of exchange effects in the reactions of quasielastic deuteron knock- out

    Energy Technology Data Exchange (ETDEWEB)

    Markov, V I; Poch, L

    1974-12-31

    A guess for the ratio of the cross sections for scattering of protons on singlet and triplet quasi-deuterons is presented in pole and triangle diagram approximations. In both cases the ratio was found to be near unity. (2 figures, 2 tables) (auth)

  19. How the Sun Knocks Out My Cell Phone from 150 Million Kilometers Away

    Science.gov (United States)

    Ladbury, Ray

    2014-01-01

    Large solar particle events (SPE) threaten many elements of critical infrastructure. A 2013 study by Lloyds of London and Atmospheric and Environmental Research recently found that if a worst-case solar event like the 1859 Carrington Event struck our planet now, it could result on $0.6-$2.36 trillion in damages to the economy. In March 2014, researchers Y. D. Liu et al. revealed that just such an event had narrowly missed Earth in July 2012. The event was observed by the STEREO A spacecraft. In this presentation, we examine how the sun can pack such a punch from 150 million km away, the threats such solar particle events pose, their mechanisms and the efforts NASA and other space agencies are carrying out to understand and mitigate such risks.

  20. Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor.

    NARCIS (Netherlands)

    Hovingh, Elise Sofie; Mariman, Rob; Solans, Luis; Hijdra, Daniëlle; Hamstra, Hendrik-Jan; Jongerius, Ilse; van Gent, Marjolein; Mooi, Frits; Locht, Camille; Pinelli, Elena

    2018-01-01

    Whooping cough, caused by Bordetella pertussis, has resurged and presents a global health burden worldwide. B. pertussis strains unable to produce the acellular pertussis vaccine component pertactin (Prn), have been emerging and in some countries represent up to 95% of recent clinical isolates.

  1. Mutated but Not Deleted Ovine PrP(C) N-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice.

    Science.gov (United States)

    Khalifé, Manal; Reine, Fabienne; Paquet-Fifield, Sophie; Castille, Johan; Herzog, Laetitia; Vilotte, Marthe; Moudjou, Mohammed; Moazami-Goudarzi, Katayoun; Makhzami, Samira; Passet, Bruno; Andréoletti, Olivier; Vilette, Didier; Laude, Hubert; Béringue, Vincent; Vilotte, Jean-Luc

    2016-02-01

    Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease. Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. A DTAP–IPV//PRP~T VACCINE: A REVIEW OF 16 YEARS’ CLINICAL EXPERIENCE

    Directory of Open Access Journals (Sweden)

    Stanley A. Plotkin

    2012-01-01

    Full Text Available Owing to their low reactogenicity, confirmed efficacy and availability in combination vaccines, acellular pertussis (aP-inactivated poliovirus (IPV combined vaccines are now included in various national immunization programs worldwide. We provide an overview of 16 years of clinical experience with a diphtheria (D, tetanus (T, aP, IPV and Haemophilus influenzae type b (Hib polysaccharide conjugated to tetanus protein (PRP~T combined vaccine (DTaP–IPV//PRP~T — Pentaxim, Sanofi Pasteur, France. Good immunogenicity has been demonstrated after primary vaccination with Pentaxim, regardless of the population ethnicity and primary vaccination schedule. A booster vaccination in the second year of life also resulted in a high immune response for each antigen. Furthermore, 10 years of national surveillance in Sweden has demonstrated the effectiveness of Pentaxim in controlling pertussis. As is the case for other aP-containing combined vaccines, Pentaxim is well tolerated, with the safety profile being better than for whole-cell pertussiscontaining combination vaccines for primary and booster vaccinations.

  3. Prediction and optimization of the recovery rate in centrifugal separation of platelet-rich plasma (PRP)

    Science.gov (United States)

    Piao, Linfeng; Park, Hyungmin; Jo, Chris

    2016-11-01

    We present a theoretical model of the recovery rate of platelet and white blood cell in the process of centrifugal separation of platelet-rich plasma (PRP). For the practically used conditions in the field, the separation process is modeled as a one-dimensional particle sedimentation; a quasi-linear partial differential equation is derived based on the kinematic-wave theory. This is solved to determine the interface positions between supernatant-suspension and suspension-sediment, used to estimate the recovery rate of the plasma. While correcting the Brown's hypothesis (1989) claiming that the platelet recovery is linearly proportional to that of plasma, we propose a new correlation model for prediction of the platelet recovery, which is a function of the volume of whole blood, centrifugal acceleration and time. For a range of practical parameters, such as hematocrit, volume of whole blood and centrifugation (time and acceleration), the predicted recovery rate shows a good agreement with available clinical data. We propose that this model is further used to optimize the preparation method of PRP that satisfies the customized case. Supported by a Grant (MPSS-CG-2016-02) through the Disaster and Safety Management Institute funded by Ministry of Public Safety and Security of Korean government.

  4. CLINICAL RESULTS FROM THE TREATMENT OF CHRONIC SKIN WOUNDS WITH PLATELET RICH PLASMA (PRP

    Directory of Open Access Journals (Sweden)

    Pencho Kossev

    2015-12-01

    Full Text Available PURPOSE: To show platelet rich plasma (PRP application of chronic skin wounds and to evaluate the results from the treatment. MATERIAL AND METHODS: A total of 14 patients with problematic skin wounds had been treated at the clinic for a period of five years (from May 2009 to December 2014 with the following patient sex ratio: male patients - 5 and female patients - 9. Average age - 48,5 (30-76. Patients with Type 2 Diabetes - 4, with decubitus ulcers - 6, traumatic - 8, with infection - 5. Based on a scheme developed by us, all cases were treated by administering platelet-rich plasma, derived by PRGF Endoret system. Follow-up period was within 4 - 6 months (4,5 on average. RESULTS: The results have been evaluated based on the following functional scoring systems - Total wound score, Total anatomic score and Total score (20. The baseline values at the very beginning of the follow-up period were as follows: Total wound score - 12 p.; Total anatomic score - 10 p., Total score - 17 p. By the end of the treatment period the score was 0 p., which means excellent results, i.e. complete healing of the wounds. CONCLUSION: We believe that the application of PRP may become optimal therapy in the treatment of difficult to heal wounds around joints, bone, subject tendons, plantar surface of the foot, etc., as it opens new perspectives in the field of human tissue regeneration.

  5. PRP Comments for ICF Q1/Q2 FY17 Experiments 3/10/16

    Energy Technology Data Exchange (ETDEWEB)

    Kauffman, R. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-04-14

    The PRP generally endorsed the Program plan during the short time for discussions. We agree that the strategy to develop a hohlraum that is symmetric and has low laser-plasma instabilities and to develop an alternative method for supporting the capsule is the best path forward for making progress in understanding ignition performance. The Program is oriented toward a milestone in 2020 for “determining the efficacy of NIF for ignition and credible physics-scaling to multi-megajoule yields for all ICF approaches.” We are concerned that the time and resources are not sufficient to vet all of the various approaches that are being pursued to make an informed decision by this date. For NIF to meet this goal, a process will be needed to to select the most promising paths forward. We recommend that the Program develop this process for selecting the path forward to optimize resources. We were glad to see that the direct drive program took our comments under consideration. We think that the proposed experiments have the program headed in a better direction. The PRP had only a short time to discuss the detailed experimental proposals. The following are comments on the detailed proposals. We did not have time to discuss them as a group. They represent individual opinions and provided to you as feedback to your proposals.

  6. A modified three-term PRP conjugate gradient algorithm for optimization models.

    Science.gov (United States)

    Wu, Yanlin

    2017-01-01

    The nonlinear conjugate gradient (CG) algorithm is a very effective method for optimization, especially for large-scale problems, because of its low memory requirement and simplicity. Zhang et al. (IMA J. Numer. Anal. 26:629-649, 2006) firstly propose a three-term CG algorithm based on the well known Polak-Ribière-Polyak (PRP) formula for unconstrained optimization, where their method has the sufficient descent property without any line search technique. They proved the global convergence of the Armijo line search but this fails for the Wolfe line search technique. Inspired by their method, we will make a further study and give a modified three-term PRP CG algorithm. The presented method possesses the following features: (1) The sufficient descent property also holds without any line search technique; (2) the trust region property of the search direction is automatically satisfied; (3) the steplengh is bounded from below; (4) the global convergence will be established under the Wolfe line search. Numerical results show that the new algorithm is more effective than that of the normal method.

  7. Knee Osteoarthritis Injection Choices: Platelet- Rich Plasma (PRP versus Hyaluronic Acid (A one-year randomized clinical trial

    Directory of Open Access Journals (Sweden)

    Seyed Ahmad Raeissadat

    2015-01-01

    Full Text Available Introduction Knee osteoarthritis (OA is the most common articular disease. Different methods are used to alleviate the symptoms of patients with knee OA, including analgesics, physical therapy, exercise prescription, and intra-articular injections (glucocorticoids, hyaluronic acid [HA], etc. New studies have focused on modern therapeutic methods that stimulate cartilage healing process and improve the damage, including the use of platelet-rich plasma (PRP as a complex of growth factors. Due to the high incidence of OA and its consequences, we decided to study the long-term effect of intraarticular injection of PRP and HA on clinical outcome and quality of life of patients with knee OA. Method This non-placebo-controlled randomized clinical trial involved 160 patients affected by knee OA, grade 1–4 of Kellgren–Lawrence scale. In the PRP group ( n = 87, two intra-articular injections at 4-week interval were applied, and in the HA group ( n = 73, three doses of intra-articular injection at 1-week interval were applied. All patients were prospectively evaluated before and at 12 months after the treatment by Western Ontario and McMaster Universities Arthritis Index (WOMAC and SF-36 questionnaires. The results were analyzed using SPSS 16.1 software (RCT code: IRCT2014012113442N5. Results At the 12-month follow-up, WOMAC pain score and bodily pain significantly improved in both groups; however, better results were determined in the PRP group compared to the HA group ( P < 0.001. Other WOMAC and SF-36 parameters improved only in the PRP group. More improvement (but not statistically significant was achieved in patients with grade 2 OA in both the groups. Conclusion This study suggests that PRP injection is more efficacious than HA injection in reducing symptoms and improving quality of life and is a therapeutic option in select patients with knee OA who have not responded to conventional treatment.

  8. On the extent of homogeneity region of PrP phase in the system praseodymium-phosphorus

    International Nuclear Information System (INIS)

    Mironov, K.E.

    1984-01-01

    For constructed by ion type compounds in the metal or metalloid systems homogeneity region boundary position can be observed at different compositions depending on which side the approximation to it occurs: on the metal or compound side. As an example the PrP homogeneity region in the praseodymium-phosphorus system is considered. An assumption is made on the prevalence of this phenomenon among rare earth monopnictides and monochalcogenides. For the PrP phase it is indicated that the monophopshide cell parameter depends on content of impurities in the initial metal, oxygen, in particular

  9. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Moussa, Mayssam, E-mail: Moussa-mayssam@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Lajeunesse, Daniel, E-mail: daniel.lajeunesse@umontreal.ca [Research Centre in Osteoarthritis, Research Centre in Monteral University (Canada); Hilal, George, E-mail: George2266@gmail.com [Cancer and metabolism Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); El Atat, Oula, E-mail: oulaatat@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Haykal, Gaby, E-mail: Gaby.haykal@hdf.usj.edu.lb [Hotel Dieu de France, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Serhal, Rim, E-mail: rim.basbous@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Chalhoub, Antonio, E-mail: Mava.o@hotmail.com [Carantina Hospital, Beirut (Lebanon); Khalil, Charbel, E-mail: charbelk3@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Alaaeddine, Nada, E-mail: Nada.aladdin@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon)

    2017-03-01

    Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1–2–3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. - Highlights: • Platelet Rich Plasma is suggested as a new treatment for osteoarthritis. • The proposed therapeutic effect is

  10. Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106-126

    DEFF Research Database (Denmark)

    Bergstrøm, Linda Alice; Chabry, J.; Bastholm, L.

    2007-01-01

    There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation...... tendency and neurotoxicity of different molecular variants of the prion peptide PrP106-126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity...

  11. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

    International Nuclear Information System (INIS)

    Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

    2017-01-01

    Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1–2–3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. - Highlights: • Platelet Rich Plasma is suggested as a new treatment for osteoarthritis. • The proposed therapeutic effect is

  12. PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.

    Science.gov (United States)

    Maréchal, Alexandre; Li, Ju-Mei; Ji, Xiao Ye; Wu, Ching-Shyi; Yazinski, Stephanie A; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E; Jin, Jianping; Zou, Lee

    2014-01-23

    PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. A Comparison of Platelet Count and Enrichment Percentages in the Platelet Rich Plasma (PRP) Obtained Following Preparation by Three Different Methods.

    Science.gov (United States)

    Sabarish, Ram; Lavu, Vamsi; Rao, Suresh Ranga

    2015-02-01

    Platelet rich plasma (PRP) represents an easily accessible and rich source of autologous growth factors. Different manual methods for the preparation of PRP have been suggested. Lacuna in knowledge exists about the efficacy of PRP preparation by these different manual methods. This study was performed to determine the effects of centrifugation rate revolutions per minute (RPM) and time on the platelet count and enrichment percentages in the concentrates obtained following the three different manual methods of PRP preparation. In vitro experimental study. This was an experimental study in which platelet concentration was assessed in the PRP prepared by three different protocols as suggested by Marx R (method 1), Okuda K (method 2) and Landesberg R (method 3). A total of 60 peripheral blood samples, (n=20 per method) were obtained from healthy volunteers. Baseline platelet count was assessed for all the subjects following which PRP was prepared. The platelet count in the PRP was determined using coulter counter (Sysmex XT 2000i). The mean of the platelet count obtained and their enrichment percentage were calculated and intergroup comparison was done (Tukey's HSD test). The number of platelets and enrichment percentage in PRP prepared by method 1 was higher compared to method 2 and method 3; this difference in platelet concentrates was found to be statistically significant (p < 0.05). The centrifugation rate and time appear to be important parameters, which influence the platelet yield. Method 1 which had lower centrifugation rate and time yielded a greater platelet count and enrichment percentage.

  14. The application of PRP combined with TCP in repairing avascular necrosis of the femoral head after femoral neck fracture in rabbit.

    Science.gov (United States)

    Zhang, X-L; Wang, Y-M; Chu, K; Wang, Z-H; Liu, Y-H; Jiang, L-H; Chen, X; Zhou, Z-Y; Yin, G

    2018-02-01

    In view of the high occurrence of avascular necrosis of the femoral head (ANFH) after femoral neck fracture and the difficulties in the treatment, our work aimed to explore the effects of platelet-rich plasma (PRP) combined with tri-calcium phosphate (TCP) on the repair of ANFH after femoral neck fracture and to provide reference for clinical treatment. Thirty New Zealand white rabbits were randomly divided into control group, TCP group, and PRP+TCP group. The rabbit ANFH model was established and femoral head tissues were collected. HE staining was used for histological observation. Image analysis and statistical analysis were used to calculate the New Bone Area fraction (NBA %). The levels of bone morphogenetic protein (BMP)-7, transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), interleukin (IL)-6 and tumor necrosis factor (TNF)-a in serum were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). The new bone area of TCP group was significantly lower than that of PRP+TCP group (pPRP+TCP groups (pPRP+TCP group was higher than that in TCP group. TCP and PRP+TCP can both significantly reduce the content of IL-6 and TNF-a (pPRP+TCP group compared with the TCP group at 8 weeks after injection. PRP combined with TCP, which can promote new bone formation and inhibit inflammatory response, showed higher efficiency in repairing ANFH than internal fixation alone.

  15. Investigation of modified platelet-rich plasma (mPRP in promoting the proliferation and differentiation of dental pulp stem cells from deciduous teeth

    Directory of Open Access Journals (Sweden)

    J. Wen

    2016-01-01

    Full Text Available Stem cells from human exfoliated deciduous teeth (SHEDs have great potential to treat various dental-related diseases in regenerative medicine. They are usually maintained with 10% fetal bovine serum (FBS in vitro. Modified platelet-rich plasma (mPRP would be a safe alternative to 10% FBS during SHEDs culture. Therefore, our study aimed to compare the proliferation and differentiation of SHEDs cultured in mPRP and FBS medium to explore an optimal concentration of mPRP for SHEDs maintenance. Platelets were harvested by automatic blood cell analyzer and activated by repeated liquid nitrogen freezing and thawing. The platelet-related cytokines were examined and analyzed by ELISA. SHEDs were extracted and cultured with different concentrations of mPRP or 10% FBS medium. Alkaline phosphatase (ALP activity was measured. Mineralization factors, RUNX2 and OCN, were measured by real-time PCR. SHEDs were characterized with mesenchymal stem cells (MSCs markers including vimentin, CD44, and CD105. mPRP at different concentrations (2, 5, 10, and 20% enhanced the growth of SHEDs. Moreover, mPRP significantly stimulated ALP activity and promoted expression of RUNX2 and OCN compared with 10% FBS. mPRP could efficiently facilitate proliferation and differentiation of SHEDs, and 2% mPRP would be an optimal substitute for 10% FBS during SHEDs expansion and differentiation in clinical scale manufacturing.

  16. Rasgrf2 controls dopaminergic adaptations to alcohol in mice.

    Science.gov (United States)

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

    2014-10-01

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

  17. Evaluation of Not-Activated and Activated PRP in Hair Loss Treatment: Role of Growth Factor and Cytokine Concentrations Obtained by Different Collection Systems.

    Science.gov (United States)

    Gentile, Pietro; Cole, John P; Cole, Megan A; Garcovich, Simone; Bielli, Alessandra; Scioli, Maria Giovanna; Orlandi, Augusto; Insalaco, Chiara; Cervelli, Valerio

    2017-02-14

    Platelet rich plasma (PRP) was tested as a potential therapy for androgenetic alopecia (AGA) through two different clinical protocols in which one population (18 participants) received half-head treatment with autologous non-activated PRP (A-PRP) produced by CPunT Preparation System (Biomed Device, Modena, Italy) and the other half-head with placebo, and a second separated population in which all participants (n = 6, 3 participants per group) received treatment with calcium-activated PRP (AA-PRP) produced from one of two different PRP collection devices (Regen Blood Cell Therapy or Arthrex Angel System). For the A-PRP study, three treatments were administered over 30-day intervals. Trichoscan analysis of patients, three months post-treatment, showed a clinical improvement in the number of hairs in the target area (36 ± 3 hairs) and in total hair density (65±  5 hair cm2), whereas negligible improvements in hair count (1.1±  1.4 hairs) and density (1.9 ± 10.2 hair cm2) were seen in the region of the scalp that received placebo. Microscopic evaluation conducted two weeks after treatment showed also an increase in epidermal thickness, Ki67+ keratinocytes, and in the number of follicles. The AA-PRP treatment groups received a singular set of injections, and six months after the treatments were administered, notable differences in clinical outcomes were obtained from the two PRP collection devices (+90 ± 6 hair cm2 versus -73 ± 30 hair cm2 hair densities, Regen versus Arthrex). Growth factor concentrations in AA-PRP prepared from the two collection devices did not differ significantly upon calcium activation.

  18. Evaluation of Not-Activated and Activated PRP in Hair Loss Treatment: Role of Growth Factor and Cytokine Concentrations Obtained by Different Collection Systems

    Directory of Open Access Journals (Sweden)

    Pietro Gentile

    2017-02-01

    Full Text Available Platelet rich plasma (PRP was tested as a potential therapy for androgenetic alopecia (AGA through two different clinical protocols in which one population (18 participants received half-head treatment with autologous non-activated PRP (A-PRP produced by CPunT Preparation System (Biomed Device, Modena, Italy and the other half-head with placebo, and a second separated population in which all participants (n = 6, 3 participants per group received treatment with calcium-activated PRP (AA-PRP produced from one of two different PRP collection devices (Regen Blood Cell Therapy or Arthrex Angel System. For the A-PRP study, three treatments were administered over 30-day intervals. Trichoscan analysis of patients, three months post-treatment, showed a clinical improvement in the number of hairs in the target area (36 ± 3 hairs and in total hair density (65±  5 hair cm2, whereas negligible improvements in hair count (1.1±  1.4 hairs and density (1.9 ± 10.2 hair cm2 were seen in the region of the scalp that received placebo. Microscopic evaluation conducted two weeks after treatment showed also an increase in epidermal thickness, Ki67+ keratinocytes, and in the number of follicles. The AA-PRP treatment groups received a singular set of injections, and six months after the treatments were administered, notable differences in clinical outcomes were obtained from the two PRP collection devices (+90 ± 6 hair cm2 versus -73 ± 30 hair cm2 hair densities, Regen versus Arthrex. Growth factor concentrations in AA-PRP prepared from the two collection devices did not differ significantly upon calcium activation.

  19. A Randomized, Controlled Study of DTaP-IPV-HB-PRP-T, a Fully Liquid Hexavalent Vaccine, Administered in a 3-, 5- and 11- to 12-month Schedule.

    Science.gov (United States)

    Vesikari, Timo; Silfverdal, Sven-Arne; Jordanov, Emilia; Feroldi, Emmanuel

    2017-01-01

    To assess the immunogenicity and safety of a fully liquid, ready-to-use hexavalent DTaP-IPV-HB-PRP-T vaccine when administered in a 2 + 1 schedule at 3, 5 and 11-12 months of age. Phase III, randomized, active-controlled, observer-blind, multicenter study. Infants were randomized to receive DTaP-IPV-HB-PRP-T (N = 275) or a licensed control hexavalent vaccine (DTaP-IPV-HB//PRP~T: N = 275), both given in coadministration with Prevenar 13. Serum was analyzed for immune responses to all vaccine antigens. Noninferiority of DTaP-IPV-HB-PRP-T to the control vaccine was tested at completion of the primary series using predefined seroprotection (SP) rate and vaccine response (VR) rates. Safety was assessed using parental reports. Noninferiority of DTaP-IPV-HB-PRP-T to the control vaccine was demonstrated postdose 3 for each antigen, and the SP (for D, T, poliovirus 1, 2 and 3, hepatitis B and polyribosylribitol phosphate) and VR rates (for pertussis toxin and filamentous hemagglutinin) were high in each group. SP rates for D, T, polio 1, 2, 3 and VR rates for pertussis toxin and filamentous hemagglutinin were similar in each group. For hepatitis B, SP rate was slightly higher for DTaP-IPV-HB//PRP~T (99.6%) than DTaP-IPV-HB-PRP-T (96.4%), and for PRP, SP rate was higher for DTaP-IPV-HB-PRP-T (93.5%) than DTaP-IPV-HB//PRP~T (85.2%). For Prevenar 13, the SP rate was high for each serotype and similar for both groups. All vaccines were well tolerated. These study findings confirm the safety and immunogenicity and thus the suitability of this fully liquid hexavalent vaccine for administration in a 2 + 1 schedule.

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

    Science.gov (United States)

    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

  1. Microbicidal properties of Leukocyte- and Platelet-Rich Plasma/Fibrin (L-PRP/L-PRF): new perspectives.

    Science.gov (United States)

    Cieslik-Bielecka, A; Dohan Ehrenfest, D M; Lubkowska, A; Bielecki, T

    2012-01-01

    Platelets, as main actors of the first stage of the healing process, play an important role in tissue repair. Their granules contain many active substances, particularly over 30 growth factors with significant effects on the resident cells at the site of injury, such as mesenchymal stem cells, chondrocytes, fibroblasts, osteoblasts. This potential may be increased by the concentration of the platelets, using platelet-rich plasma/fibrin products. In the four families of platelet concentrates, 2 families contain also significant concentrations of leukocytes: L-PRP (Leukocyte- and Platelet-Rich Plasma) and L-PRF (Leukocyte- and Platelet-Rich Fibrin). Inductive properties of platelet concentrates were widely described. However, they present also antimicrobial effects. The antibacterial effects of L-PRP were highlighted in only a few in vitro studies. Strong activity comparable to gentamicin and oxacillin for L-PRP against methicillin susceptible Staphylococcus aureus (MSSA) was already demonstrated. L-PRP also inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli. Some authors also reported clinical observations about the reduction of infections and the induction of healing processes after the use of platelet concentrates in cardiac, orthopaedic, oral and maxillofacial surgery. However, very little is yet known about the antibacterial effects of these concentrates. In this manuscript, the current data about the antimicrobial agents and cells present in the platelet-rich plasma/fibrin are highlighted and discussed, in order to introduce this new key chapter of the platelet concentrate technology history.

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

    Directory of Open Access Journals (Sweden)

    Molloy Mark P

    2011-06-01

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

  3. Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro.

    Science.gov (United States)

    Workman, Rob G; Maddison, Ben C; Gough, Kevin C

    2017-07-04

    Prion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.

  4. Histomorphometric Parameters of the Growth Plate and Trabecular Bone in Wild-Type and Trefoil Factor Family 3 (Tff3)-Deficient Mice Analyzed by Free and Open-Source Image Processing Software.

    Science.gov (United States)

    Bijelić, Nikola; Belovari, Tatjana; Stolnik, Dunja; Lovrić, Ivana; Baus Lončar, Mirela

    2017-08-01

    Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.

  5. Functional recovery of regenerating motor axons is delayed in mice heterozygously deficient for the myelin protein P(0) gene

    DEFF Research Database (Denmark)

    Rosberg, Mette Romer; Alvarez, Susana; Krarup, Christian

    2013-01-01

    Mice with a heterozygous knock-out of the myelin protein P0 gene (P0+/-) develop a neuropathy similar to human Charcot-Marie-Tooth disease. They are indistinguishable from wild-types (WT) at birth and develop a slowly progressing demyelinating neuropathy. The aim of this study was to investigate...... whether the regeneration capacity of early symptomatic P0+/- is impaired as compared to age matched WT. Right sciatic nerves were lesioned at the thigh in 7-8 months old mice. Tibial motor axons at ankle were investigated by conventional motor conduction studies and axon excitability studies using...... threshold tracking. To evaluate regeneration we monitored the recovery of motor function after crush, and then compared the fiber distribution by histology. The overall motor performance was investigated using Rotor-Rod. P0+/- had reduced compound motor action potential amplitudes and thinner myelinated...

  6. PLATELET-RICH PLASMA (PRP FOR THE TREATMENT OF PROBLEMATIC SKIN WOUNDS

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    Tsvetan Sokolov

    2016-12-01

    Full Text Available OBJECTIVE: To show platelet-rich plasma (PRP application of problematic skin wounds and to evaluate the results from the treatment. MATERIAL AND METHODS: A total of 31 patients with problematic skin wounds had been treated at the clinic for a period of five years (from May 2010 to September 2015 with the following patient sex ratio: male patients– 13 and female patients– 18. Average age– 46,5 (22-82. Patients with Type 2 Diabetes– 10, with decubitus ulcers– 2, traumatic– 29, with infection– 12, acute– 15, chronic– 16. Based on a scheme developed by us, all cases were treated by administering platelet-rich plasma, derived by PRGF Endoret system. Follow-up period was within 4 – 6 months (4,5 on average. We used platelet rich plasma derived by PRGF Endoret system, applied on the wound bed on a weekly basis. RESULTS: The results have been evaluated based on the following functional scoring systems - Total wound score, Total anatomic score and Total score (20. The baseline values at the very beginning of the follow-up period were as follows: Total wound score – 10 p.; Total anatomic score – 8 p., Total score – 15 p. By the end of the treatment period the score was 0 p., which means excellent results, i.e. complete healing of the wounds. CONCLUSION: We believe that the application of PRP may become optimal therapy in the treatment of difficult to heal wounds around joints, bone, subject tendons, plantar surface of the foot, etc., as it opens new perspectives in the field of human tissue regeneration.

  7. The Effect of Platelet-Rich Plasma (PRP on Improvement in Pain and Symptoms of Shoulder Subacromial Impingement Syndrome

    Directory of Open Access Journals (Sweden)

    Parisa Nejati

    2015-08-01

    Full Text Available Abstract Background: Subacromial impingement is one of the most common complaints of shoulder. Treatments include avoiding of painful activities, oral anti-pain drugs, physical therapy modalities, corticosteroid injection and exercise therapy. Some studies have shown that platelet- rich plasma(PRP is effective on tendinitis and tearing of tendons, ligaments and muscles, but evidence that has proved PRP as a conservative treatment in shoulder pathologies is very limited. This study aims to investigate the effect of PRP injection on relieving pain and improving daily function of patients with shoulder impingement syndrome. Materials and Methods: In this clinical trial study, patients older than 40 with pain more than three months were included. If they had three of four positive diagnostic clinical tests of shoulder impingement that were confirmed by shoulder MRI, could be injected PRP twice. The time between injections was 1 month. Pain was measured by visual analog scale (VAS and function was measured by two questionnaires named disabilities of the arm, shoulder and hand (DASH and western Ontario rotator cuff index (WORC. Range of motion (ROM of shoulder was measured in five directions by goniometry . All of these parameters were evaluated before intervention and in 1, 3, 6 months later. Results: with due attention to a six-month folloe-up, PRR injection was effective in pain reduction and improvement of patient's function (p<0.05. Shoulder Rom increased in all directions except external rotation and the power of shoulder muscles was evidently improved statistically in flexion, abduction and internal toration. Conclusion: PRP injection could effectively reduce pain and improve daily activities in patients with shoulder impingement syndrome.

  8. Adjunctive Platelet-Rich Plasma (PRP in Infrabony Regenerative Treatment: A Systematic Review and RCT’s Meta-Analysis

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    Mubashir Saleem

    2018-01-01

    Full Text Available Background and Objective. The purpose of this study was to highlight the clinical performance of platelet-rich plasma (PRP used as an adjunctive tool for regeneration in infrabony periodontal defects using different biomaterials or performing different surgical flap approaches. Comparative evaluation of main clinical outcomes as probing pocket depth reduction, clinical attachment gain, and recession reduction with and without the use of PRP has been analysed. Materials and Methods. According to the focused question, an electronic and hand searching has been performed up to December 2016. From a batch of 73 articles, the selection strategy and Jadad quality assessment led us to include 15 studies for the meta-analysis. Results. Despite the high heterogeneity found and the lack of complete data regarding the selected clinical outcomes, a comparative analysis has been possible by the categorization of used biomaterials and surgical flap approaches. This method led us to observe the best performance of grafts with the use of adjunctive PRP in CAL gain and PPD reduction. No difference has been outlined with a specific surgical flap. Conclusions. Although PRP is considered a cheap and patient’s derived growth factor, the not conclusive data reported would suggest that its use in addition to bone substitutes could be of some clinical benefit in the regenerative treatment of infrabony defects. Clinical Relevance. This systematic review was intended to sort out the huge controversial debate in the field about the possible use of PRP in regenerative surgery in infrabony defect. The clinical relevance of using blood-borne growth factors to conventional procedures is effective as these could determine a better performance and outcomes despite the surgical approach adopted and limit the use of additional biomaterials for the blood clot stabilization.

  9. CONTAMINATED PROBLEMATIC SKIN WOUNDS IN DIABETIC PATIENTS TREATED WITH AUTOLOGOUS PLATELET-RICH PLASMA (PRP: A case series study

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    Tsvetan Sokolov

    2016-03-01

    Full Text Available OBJECTIVE: To study the effect of platelet-rich plasma (PRP on contaminated problematic skin ulcers in patients with diabetes. MATERIAL AND METHODS: A total of 6 patients had been treated within the period from 2012 to 2014; they had various types of problematic wounds and diabetes type 2. Patients’ distribution by sex was as follows: 1 man and 5 women; mean age- 68 years. Ulcer types: acute (2 patients, hard-to-heal (2 patients and chronic (2 patients ulcers. The mean size of the skin and soft tissue defect was 9,5 cm2. Pathogenic microflora was isolated in 4 patients - S. aureus in three and Е. Coli in one. Based on a scheme developed by us, all cases were treated by administering platelet-rich plasma, derived by PRGF Endoret system. Follow-up period was within 4 – 6 months (4,5 on average. We used platelet rich plasma derived by PRGF Endoret system, applied on the wound bed on a weekly basis. RESULTS: Application of PRP allowed successful closure of all wounds. There were no complications associated with treatment of PRP. Epithelialization of the wound took 15 weeks on average for all patients. One patient presented with hyperkeratosis. Initial score of followed wounds, based on the scales are as follows: Total wound score – 10 p. Total anatomic score – 8 p. Total score – 15 p. at the initial stage. At the end of the treatment period scores were as follows - 0 p., which means excellent results CONCLUSION: We believe that the application of PRP may become optimal therapy in the treatment of contaminated problematic wounds in diabetic patients. PRP not only stimulates wound healing, but also has antimicrobial properties, which may contribute to the prevention of infections.

  10. Chemokine-like receptor 1 deficiency does not affect the development of insulin resistance and nonalcoholic fatty liver disease in mice.

    Directory of Open Access Journals (Sweden)

    Nanda Gruben

    Full Text Available The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1, are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD, which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH. It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1-/- mice and wild type (WT mice into low-density lipoprotein receptor knock-out (Ldlr-/- mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS. Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.

  11. Comparison between Conventional Mechanical Fixation and Use of Autologous Platelet Rich Plasma (PRP) in Wound Beds Prior to Resurfacing with Split Thickness Skin Graft.

    Science.gov (United States)

    P Waiker, Veena; Shivalingappa, Shanthakumar

    2015-01-01

    Platelet rich plasma is known for its hemostatic, adhesive and healing properties in view of the multiple growth factors released from the platelets to the site of wound. The primary objective of this study was to use autologous platelet rich plasma (PRP) in wound beds for anchorage of skin grafts instead of conventional methods like sutures, staplers or glue. In a single center based randomized controlled prospective study of nine months duration, 200 patients with wounds were divided into two equal groups. Autologous PRP was applied on wound beds in PRP group and conventional methods like staples/sutures used to anchor the skin grafts in a control group. Instant graft adherence to wound bed was statistically significant in the PRP group. Time of first post-graft inspection was delayed, and hematoma, graft edema, discharge from graft site, frequency of dressings and duration of stay in plastic surgery unit were significantly less in the PRP group. Autologous PRP ensured instant skin graft adherence to wound bed in comparison to conventional methods of anchorage. Hence, we recommend the use of autologous PRP routinely on wounds prior to resurfacing to ensure the benefits of early healing.

  12. Autologous platelet-rich plasma (PRP) in chronic penile lichen sclerosus: the impact on tissue repair and patient quality of life.

    Science.gov (United States)

    Casabona, Francesco; Gambelli, Ilaria; Casabona, Federica; Santi, Pierluigi; Santori, Gregorio; Baldelli, Ilaria

    2017-04-01

    Lichen sclerosus (LS) is a chronic inflammatory skin condition that frequently involves the anogenital region. Ongoing research is focused on finding more effective treatments for tissue repair and reducing symptoms. The aim of this study is to evaluate the effectiveness of platelet-rich plasma (PRP) local injections in penile LS. Forty-five male patients affected by penile LS underwent injections of autologous PRP in the affected skin areas. Age at diagnosis and at first treatment, number of treatments, clinical conditions (phimosis, splitting, inflammation, synechiae, meatus stenosis), symptoms (pain, burning, itching), and functional impairment were considered. Treatment efficacy was also evaluated through the Investigator's Global Assessment (IGA) on a six-point Likert scale and the Dermatology Life Quality Index (DLQI). The patient age at LS diagnosis was 36.20 ± 9.19 years, while the mean age at the first PRP treatment was 42.96 ± 11.32 years (p PRP injections, it was observed in all patients a significant improvement in clinical conditions, with reduction/disappearance of symptoms. Topical steroid therapy, interrupted before PRP treatment, was not restarted by any patient. Only one patient underwent a later circumcision procedure. Both IGA scale and DLQI score showed a significant difference (p PRP treatment. PRP treatment in penile LS seems to be helpful to regenerate scarring, reduce symptoms, and improve patient quality of life. Further studies are necessary to evaluate long-term results.

  13. Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

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    Shan Lu

    Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

  14. A fully liquid DTaP-IPV-HB-PRP-T hexavalent vaccine for primary and booster vaccination of healthy Turkish infants and toddlers

    Science.gov (United States)

    Ceyhan, Mehmet; Yıldırım, İnci; Tezer, Hasan; Devrim, İlker; Feroldi, Emmanuel

    2017-08-23

    Background/aim: Immunogenicity and safety of a primary series of a fully liquid, hexavalent DTaP-IPV-HB-PRP-T vaccine given at 2, 3, and 4 months of age compared to licensed comparators and a DTaP-IPV-HB-PRP-T booster at 15?18 months were evaluated. Materials and methods: This was a Phase III, randomized, open-label trial. Primary series (no hepatitis B [HB] at birth) of DTaP-IPV-HB-PRP-T (N = 155) (group 1) or licensed control vaccines (DTaP-IPV//PRP-T and standalone HB: N = 155) (group 2) and DTaP-IPV-HB-PRP-T booster were administered. Noninferiority was evaluated 1 month postprimary series for anti-HB seroprotection (SP). All other analyses were descriptive. Safety was assessed from parental reports. Results: Postprimary series noninferiority of anti-HB ≥ 10 mIU/mL was demonstrated for the DTaP-IPV-HB-PRP-T vaccine (94.0%) compared to the licensed control (96.1%). Postprimary series primary SP and seroconversion (SC) rates were high and similar for both groups. Antibody persistence (prebooster) was high for each antigen and similar between groups except for HB, which was lower for DTaP-IPV-HB-PRP-T than for standalone HB. For each antigen except HB, DTaP-IPV-HB-PRP-T booster responses were high and similar in each group. Safety was good for primary and booster series and similar between groups. Conclusion: The DTaP-IPV-HB-PRP-T vaccine is immunogenic and safe when administered in a challenging primary series schedule without HB vaccination at birth.

  15. Dissociation of frontotemporal dementia-related deficits and neuroinflammation in progranulin haploinsufficient mice.

    Science.gov (United States)

    Filiano, Anthony J; Martens, Lauren Herl; Young, Allen H; Warmus, Brian A; Zhou, Ping; Diaz-Ramirez, Grisell; Jiao, Jian; Zhang, Zhijun; Huang, Eric J; Gao, Fen-Biao; Farese, Robert V; Roberson, Erik D

    2013-03-20

    Frontotemporal dementia (FTD) is a neurodegenerative disease with hallmark deficits in social and emotional function. Heterozygous loss-of-function mutations in GRN, the progranulin gene, are a common genetic cause of the disorder, but the mechanisms by which progranulin haploinsufficiency causes neuronal dysfunction in FTD are unclear. Homozygous progranulin knock-out (Grn(-/-)) mice have been studied as a model of this disorder and show behavioral deficits and a neuroinflammatory phenotype with robust microglial activation. However, homozygous GRN mutations causing complete progranulin deficiency were recently shown to cause a different neurological disorder, neuronal ceroid lipofuscinosis, suggesting that the total absence of progranulin may have effects distinct from those of haploinsufficiency. Here, we studied progranulin heterozygous (Grn(+/-)) mice, which model progranulin haploinsufficiency. We found that Grn(+/-) mice developed age-dependent social and emotional deficits potentially relevant to FTD. However, unlike Grn(-/-) mice, behavioral deficits in Grn(+/-) mice occurred in the absence of gliosis or increased expression of tumor necrosis factor-α. Instead, we found neuronal abnormalities in the amygdala, an area of selective vulnerability in FTD, in Grn(+/-) mice. Our findings indicate that FTD-related deficits resulting from progranulin haploinsufficiency can develop in the absence of detectable gliosis and neuroinflammation, thereby dissociating microglial activation from functional deficits and suggesting an important effect of progranulin deficiency on neurons.

  16. Matrix metalloproteinase content and activity in low-platelet, low-leukocyte and high-platelet, high-leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts.

    Science.gov (United States)

    Pifer, Matthew A; Maerz, Tristan; Baker, Kevin C; Anderson, Kyle

    2014-05-01

    Recent work has shown the presence of catabolic cytokines in platelet-rich plasma (PRP), but little is known about endogenous catabolic proteases such as matrix metalloproteinases (MMPs). Hypothesis/ To quantify MMP content in 2 commercially available PRP preparation systems: Arthrex Double Syringe System autologous conditioned plasma (ACP) and Biomet GPS (GPS). The hypothesis was that MMPs are actively secreted from PRP immediately after preparation. Controlled laboratory study. PRP was prepared using either ACP (low platelet, low leukocyte) or GPS (high platelet, high leukocyte). MMP-2, MMP-3, and MMP-9 concentrations were measured using multiplex enzyme-linked immunosorbent assays for up to 6 days in 2 donors, and MMP activity was measured in 3 donors using kinetic activity kits able to detect the enzymatic cleavage of a fluorogenic peptide. Human ligament fibroblasts were cultured and exposed to both ACP and GPS from 1 donor each. MMP-2, -3, and -9 concentrations were assayed in culture media at 24 and 48 hours after exposure. GPS exhibited higher total MMP-2, -3, and -9 concentrations for up to 144 hours of release, while ACP had higher platelet-normalized MMP-2 and MMP-3 concentrations. GPS had significantly higher total and endogenous MMP-2 activity (P = .004 and .014, respectively), MMP-3 activity (P = .020 and .015, respectively), and MMP-9 activity (P = .004 and .002, respectively) compared with ACP. Once normalized to platelet count, differences in MMP activity were not significant between ACP and GPS. Compared with controls, cells stimulated with interleukin-1 beta (IL-1β) and treated with ACP showed significantly higher fold changes of MMP-2 (P = .001) and MMP-3 (P = .003) concentrations at 24 hours than did cells treated with GPS. Total MMP-9 content was higher in the media of GPS-treated, IL-1β-stimulated cells compared with ACP-treated cells (P = .001). At 48 hours, IL-1β-stimulated cells treated with GPS exhibited higher fold changes of MMP-2

  17. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

    Directory of Open Access Journals (Sweden)

    Cornelius Schneider

    Full Text Available Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

  18. Platelet-Rich Plasma (PRP) Rinses for the Treatment of Non-Responding Oral Lichen Planus: A Case Report

    OpenAIRE

    Elisabetta Merigo; Aldo Oppici; Anna Parlatore; Luigi Cella; Fabio Clini; Matteo Fontana; Carlo Fornaini

    2018-01-01

    Platelet-rich plasma (PRP) has been proposed for different applications in the medical field and in maxillofacial surgery thanks to its many growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and keratinocyte growth factor (KGF). Oral lichen planus (OLP) is a disease that affects the oral mucosa in a chronic way. This disease frequently worsens the quality of life of patients, particularly when clinical manifestations are of the erythematous or erosive/ulce...

  19. Effect of intralesional platelet-rich plasma (PRP) treatment on clinical and ultrasonographic parameters in equine naturally occurring superficial digital flexor tendinopathies - a randomized prospective controlled clinical trial.

    Science.gov (United States)

    Geburek, Florian; Gaus, Moritz; van Schie, Hans T M; Rohn, Karl; Stadler, Peter M

    2016-09-07

    Regenerative and anti-inflammatory effects on tendinopathies have been attributed to blood-derived biologicals. To date the evidence for the efficacy of autologous platelet-rich plasma (PRP) treatment of naturally occurring equine tendinopathies is limited. The purpose of this placebo-controlled clinical trial was to describe the effect of a single treatment of equine superficial digital flexor tendon (SDFT) disease with PRP on clinical and ultrasonographic parameters. Twenty horses with naturally occurring tendinopathies of forelimb SDFTs were randomly assigned to the PRP-treated group (n = 10) or control group (n = 10) after clinical and ultrasonographic examination. The SDFTs received an intralesional treatment with autologous PRP or were injected with saline, respectively (day 0). All horses participated in a standardized exercise programme and were re-examined clinically, with B-mode ultrasonography (5 times at regular intervals) and ultrasound tissue characterization (week 12 and 24 after treatment) until week 24. Long-term performance was estimated via telephone inquiry. Compared to day 0, lameness decreased significantly by week 8 after treatment with PRP and by week 12 in the control group. Ultrasonographically there was no difference in the summarized cross sectional area between the groups at any time point. Ultrasound tissue characterization showed that echo types representing disorganized matrix decreased significantly throughout the observation period in the PRP-treated group. Echo type II, representing discontinuous fascicles, not yet aligned into lines of stress was significantly higher 24 weeks after PRP treatment. Eighty percent of the PRP treated horses reached their previous or a higher level of performance after 12 months compared to 50 % in the CG. After 24 months these proportions were 60 % and 50 %, respectively. A single intralesional treatment with PRP up to 8 weeks after onset of clinical signs of tendinopathy contributes

  20. Multiple PRP injections are more effective than single injections and hyaluronic acid in knees with early osteoarthritis: a randomized, double-blind, placebo-controlled trial.

    Science.gov (United States)

    Görmeli, Gökay; Görmeli, Cemile Ayşe; Ataoglu, Baybars; Çolak, Cemil; Aslantürk, Okan; Ertem, Kadir

    2017-03-01

    To compare the effectiveness of intraarticular (IA) multiple and single platelet-rich plasma (PRP) injections as well as hyaluronic acid (HA) injections in different stages of osteoarthritis (OA) of the knee. A total of 162 patients with different stages of knee OA were randomly divided into four groups receiving 3 IA doses of PRP, one dose of PRP, one dose of HA or a saline injection (control). Then, each group was subdivided into two groups: early OA (Kellgren-Lawrence grade 0 with cartilage degeneration or grade I-III) and advanced OA (Kellgren-Lawrence grade IV). The patients were evaluated before the injection and at the 6-month follow-ups using the EuroQol visual analogue scale (EQ-VAS) and International Knee Documentation Committee (IKDC) subjective scores. Adverse events and patient satisfaction were recorded. There was a statistically significant improvement in the IKDC and EQ-VAS scores in all the treatment groups compared with the control group. The knee scores of patients treated with three PRP injections were significantly better than those patients of the other groups. There was no significant difference in the scores of patients injected with one dose of PRP or HA. In the early OA subgroups, significantly better clinical results were achieved in the patients treated with three PRP injections, but there was no significant difference in the clinical results of patients with advanced OA among the treatment groups. The clinical results of this study suggest IA PRP and HA treatment for all stages of knee OA. For patients with early OA, multiple (3) PRP injections are useful in achieving better clinical results. For patients with advanced OA, multiple injections do not significantly improve the results of patients in any group. I.

  1. Platelet-rich plasma: why intra-articular? A systematic review of preclinical studies and clinical evidence on PRP for joint degeneration.

    Science.gov (United States)

    Filardo, G; Kon, E; Roffi, A; Di Matteo, B; Merli, M L; Marcacci, M

    2015-09-01

    The aim of this review was to analyze the available evidence on the clinical application of this biological approach for the injective treatment of cartilage lesions and joint degeneration, together with preclinical studies to support the rationale for the use of platelet concentrates, to shed some light and give indications on what to treat and what to expect from intra-articular injections of platelet-rich plasma (PRP). All in vitro, in vivo preclinical and clinical studies on PRP injective treatment in the English language concerning the effect of PRP on cartilage, synovial tissue, menisci, and mesenchymal stem cells were considered. A systematic review on the PubMed database was performed using the following words: (platelet-rich plasma or PRP or platelet concentrate or platelet lysate or platelet supernatant) and (cartilage or chondrocytes or synoviocytes or menisci or mesenchymal stem cells). Fifty-nine articles met the inclusion criteria: 26 were in vitro, 9 were in vivo, 2 were both in vivo and in vitro, and 22 were clinical studies. The analysis showed an increasing number of published studies over time. Preclinical evidence supports the use of PRP injections that might promote a favourable environment for joint tissues healing. Only a few high-quality clinical trials have been published, which showed a clinical improvement limited over time and mainly documented in younger patients not affected by advanced knee degeneration. Besides the limits and sometimes controversial findings, the preclinical literature shows an overall support toward this PRP application. An intra-articular injection does not just target cartilage; instead, PRP might influence the entire joint environment, leading to a short-term clinical improvement. Many biological variables might influence the clinical outcome and have to be studied to optimize PRP injective treatment of cartilage degeneration and osteoarthritis.

  2. A fully liquid DTaP-IPV-Hep B-PRP-T hexavalent vaccine for primary and booster vaccination of healthy Mexican children.

    Science.gov (United States)

    Aquino, Amalia Guadalupe Becerra; Brito, Maricruz Gutiérrez; Doniz, Carlos E Aranza; Herrera, Juan Francisco Galán; Macias, Mercedes; Zambrano, Betzana; Plennevaux, Eric; Santos-Lima, Eduardo

    2012-10-05

    To evaluate an investigational, fully liquid hexavalent diphtheria-tetanus-acellular pertussis-inactivated poliovirus-hepatitis B-Haemophilus influenzae type b (DTaP-IPV-Hep B-PRP-T: Hexaxim™) vaccine for primary and booster vaccination of healthy children in Mexico. Infants (N=1189) were randomized to receive one of three lots of the DTaP-IPV-Hep B-PRP-T vaccine or a licensed hexavalent control vaccine (Infanrix™ hexa) for primary vaccination at 2, 4 and 6 months. All participants who completed the primary series and agreed to participate in the booster part of the study received a dose of the investigational vaccine at 15-18 months of age. Validated serological assays and parental reports were used to assess immunogenicity and safety, respectively. Post-primary vaccination, ≥95.8% of participants in both the DTaP-IPV-Hep B-PRP-T and control groups were seroprotected (SP) against diphtheria, tetanus, poliovirus, hepatitis B and PRP, or had seroconverted (SC) to the pertussis toxin (PT) and filamentous hemagglutinin (FHA) pertussis antigens. The SP/SC rates induced by the three DTaP-IPV-Hep B-PRP-T lots were equivalent. No differences in SP/SC rates were observed between the pooled lots of investigational vaccine and the control vaccine. Antibody persistence at 15-18 months was comparable between groups, with strong increases in all antibody concentrations post-DTaP-IPV-Hep B-PRP-T booster. Both vaccines were well tolerated for primary vaccination, as was the booster dose of DTaP-IPV-Hep B-PRP-T. These study findings confirm the suitability of the combined, fully liquid DTaP-IPV-Hep B-PRP-T vaccine for inclusion in routine childhood vaccination schedules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

    Science.gov (United States)

    Honda, Ryo; Kuwata, Kazuo

    2018-02-01

    Amyloid fibrils are filamentous protein aggregates associated with the pathogenesis of a wide variety of human diseases. The formation of such aggregates typically follows nucleation-dependent kinetics, wherein the assembly and structural conversion of amyloidogenic proteins into oligomeric aggregates (nuclei) is the rate-limiting step of the overall reaction. In this study, we sought to gain structural insights into the oligomeric nuclei of the human prion protein (PrP) by preparing a series of deletion mutants lacking 14-44 of the C-terminal 107 residues of PrP and examined the kinetics and thermodynamics of these mutants in amyloid formation. An analysis of the experimental data using the concepts of the Φ-value analysis indicated that the helix 2 region (residues 168-196) acquires an amyloid-like β-sheet during nucleation, whereas the other regions preserves a relatively disordered structure in the nuclei. This finding suggests that the helix 2 region serves as the nucleation site for the assembly of amyloid fibrils.-Honda, R., Kuwata, K. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

  4. Behavior of Gingival Fibroblasts on Titanium Implant Surfaces in Combination with either Injectable-PRF or PRP

    Directory of Open Access Journals (Sweden)

    Xuzhu Wang

    2017-02-01

    Full Text Available Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient’s own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF was compared to standard platelet-rich plasma (PRP with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g for 3 min were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF, transforming growth factor-β (TGF-β, collagen1 (COL1 and fibronectin (FN. The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA levels of PDGF, TGF-β, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.

  5. The Effect of Autologous Activated Platelet Rich Plasma (AA-PRP Injection on Pattern Hair Loss: Clinical and Histomorphometric Evaluation

    Directory of Open Access Journals (Sweden)

    V. Cervelli

    2014-01-01

    Full Text Available To investigate the safety and clinical efficacy of AA-PRP injections for pattern hair loss. AA-PRP, prepared from a small volume of blood, was injected on half of the selected patients’ scalps with pattern hair loss. The other half was treated with placebo. Three treatments were given for each patient, with intervals of 1 month. The endpoints were hair re-growth, hair dystrophy as measured by dermoscopy, burning or itching sensation, and cell proliferation as measured by Ki-67 evaluation. At the end of the 3 cycles of treatment, the patients presented clinical improvement in the mean number of hairs, with a mean increase of 18.0 hairs in the target area, and a mean increase in total hair density of 27.7 ( number of hairs/cm2 compared with baseline values. Microscopic evaluation showed the increase of epidermis thickness and of the number of hair follicles two weeks after the last AA-PRP treatment compared to baseline value (P<0.05. We also observed an increase of Ki67+ keratinocytes of epidermis and of hair follicular bulge cells and a slight increase of small blood vessels around hair follicles in the treated skin compared to baseline (P<0.05.

  6. No effect of ablation of surfactant protein-D on acute cerebral infarction in mice

    DEFF Research Database (Denmark)

    Lambertsen, Kate Lykke; Østergaard, Kamilla; Clausen, Bettina Hjelm

    2014-01-01

    known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory...... and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry......-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected...

  7. Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion*

    Science.gov (United States)

    Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C.; Wang, Tong

    2016-01-01

    Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk−/− mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1−/− mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1+/+ and Romk1−/− mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1+/+, but such regulation by high K intake was diminished with significant hyperkalemia in Romk1−/− mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K+ secretion in the collecting tubule. PMID:26728465

  8. Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion.

    Science.gov (United States)

    Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C; Wang, Tong

    2016-03-04

    Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Effect of Platelet-Rich Plasma (PRP versus Autologous Whole Blood on Pain and Function Improvement in Tennis Elbow: A Randomized Clinical Trial

    Directory of Open Access Journals (Sweden)

    Seyed Ahmad Raeissadat

    2014-01-01

    Full Text Available Background. Autologous whole blood and platelet-rich plasma (PRP have been both suggested to treat chronic tennis elbow. The aim of the present study was to compare the effects of PRP versus autologous whole blood local injection in chronic tennis elbow. Methods. Forty patients with tennis elbow were randomly divided into 2 groups. Group 1 was treated with a single injection of 2 mL of autologous PRP and group 2 with 2 mL of autologous blood. Tennis elbow strap, stretching, and strengthening exercises were administered for both groups during a 2-month followup. Pain and functional improvements were assessed using visual analog scale (VAS, modified Mayo Clinic performance index for the elbow, and pressure pain threshold (PPT at 0, 4, and 8 weeks. Results. All pain and functional variables including VAS, PPT, and Mayo scores improved significantly in both groups 4 weeks after injection. No statistically significant difference was noted between groups regarding pain scores in 4-week follow-up examination (P>0.05. At 8-week reevaluations, VAS and Mayo scores improved only in PRP group (P<0.05. Conclusion. PRP and autologous whole blood injections are both effective to treat chronic lateral epicondylitis. PRP might be slightly superior in 8-week followup. However, further studies are suggested to get definite conclusion.

  10. Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

    Science.gov (United States)

    Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

    2016-09-01

    The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo.

  11. Comparative assessment of prophylactic transfusions of platelet concentrates obtained by the PRP or buffy-coat methods, in patients undergoing allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Fernández-Muñoz, Hermógenes; Plaza, Eva M; Rivera-Caravaca, José Miguel; Candela, María José; Romera, Marta; De Arriba, Felipe; Lozano, María L; Vicente, Vicente; Heras, Inmaculada; Castilla-Llorente, Cristina; Rivera, José

    2018-03-27

    Whole blood-derived platelet concentrates can be obtained by the platelet-rich plasma (PRP-PCs) or the buffy-coat (BC-PCs) method. Few studies have shown that BC-PCs display lower in vitro platelet activation, but scarce information exists regarding transfusion efficacy. We have performed a retrospective study assessing platelet transfusion in patients undergoing allogeneic hematopoietic cell transplantation (AHCT) in our clinic, before and after the implementation of BC-PCs. We reviewed clinical records corresponding to 70 PRP-PCs and 86 BC-PCs prophylactic transfusions, which were performed to 55 AHCT patients. Transfusion efficacy was assessed by the 24-h post-transfusion corrected count increment (24-h CCI) and bleeding events. Clinical factors affecting transfusion outcome were also investigated. Clinical characteristics and the total number of platelet transfusions were similar among groups. Mean donor exposure was 5.8 and 5.0 in each single PRP-PCs and BC-PCs transfusion, respectively (p PRP-PCs (8.3[2.7-13.4] vs. 4.7[1.3-8.1]; p PRP-PCs transfusion (HR 4.54; 95% CI 1.72-12.01; p = 0.002). There were no differences between both groups regarding the bleeding events. In the AHCT setting, we hypothesize that BC-PCs transfusion, when compared to PRP-PCs, results in higher CCI and reduced donor exposure, but provides no significant benefit regarding bleeding outcome.

  12. Clinical-grade quality platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelet concentrates promoted expansion of mesenchymal stromal cells.

    Science.gov (United States)

    Borghese, C; Agostini, F; Durante, C; Colombatti, A; Mazzucato, M; Aldinucci, D

    2016-08-01

    The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelets as a source of growth factors for the expansion of mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl2 -activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs. © 2016 International Society of Blood Transfusion.

  13. Immunogenicity and safety of a fully liquid aluminum phosphate adjuvanted Haemophilus influenzae type b PRP-CRM197-conjugate vaccine in healthy Japanese children: A phase III, randomized, observer-blind, multicenter, parallel-group study.

    Science.gov (United States)

    Togashi, Takehiro; Mitsuya, Nodoka; Kogawara, Osamu; Sumino, Shuji; Takanami, Yohei; Sugizaki, Kayoko

    2016-08-31

    Broad use of monovalent Haemophilus influenzae type b (Hib) conjugate vaccines based on the capsular polysaccharide polyribosyl-ribitol phosphate (PRP), has significantly reduced invasive Hib disease burden in children worldwide, particularly in children aged vaccine has been widely used since the initiation of public funding programs followed by a routine vaccination designation in 2013. We compared the immunogenicity and safety of PRP conjugated to a non-toxic diphtheria toxin mutant (PRP-CRM197) vaccine with the PRP-T vaccine when administered subcutaneously to healthy Japanese children in a phase III study. Additionally, we evaluated the immunogenicity and safety profiles of a diphtheria-tetanus acellular pertussis (DTaP) combination vaccine when concomitantly administered with either PRP-CRM197 or PRP-T vaccines. The primary endpoint was the "long-term seroprotection rate", defined as the group proportion with anti-PRP antibody titers ⩾1.0μg/mL, after the primary series. Long-term seroprotection rates were 99.3% in the PRP-CRM197 group and 95.6% in the PRP-T group. The intergroup difference (PRP-CRM197 group - PRP-T group) was 3.7% (95% confidence interval: 0.099-7.336), demonstrating that PRP-CRM197 vaccine was non-inferior to PRP-T vaccine (pvaccination was higher in the PRP-CRM197 group than in PRP-T. Concomitant administration of PRP-CRM197 vaccine with DTaP vaccine showed no differences in terms of immunogenicity compared with concomitant vaccination with PRP-T vaccine and DTaP vaccine. Although CRM197 vaccine had higher local reactogenicity, overall, both Hib vaccines had acceptable safety and tolerability profiles. The immunogenicity of PRP-CRM197 vaccine administered subcutaneously as a three-dose primary series in children followed by a booster vaccination 1year after the primary series induced protective levels of Hib antibodies with no safety or tolerability concerns. Registered on ClinicalTrials.gov: NCT01379846. Copyright © 2016 The Authors

  14. Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

    Directory of Open Access Journals (Sweden)

    Emmanuel A Asante

    Full Text Available Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP gene (PRNP and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc, pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((CtmPrP. Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc was demonstrated in the brains of recipient transgenic mice. This PrP(Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

  15. Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

    Science.gov (United States)

    Asante, Emmanuel A; Linehan, Jacqueline M; Smidak, Michelle; Tomlinson, Andrew; Grimshaw, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Powell, Caroline; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

    2013-01-01

    Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc)), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((Ctm)PrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc) was demonstrated in the brains of recipient transgenic mice. This PrP(Sc) rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (Ctm)PrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-08-10

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

  17. The folding mechanism and key metastable state identification of the PrP127-147 monomer studied by molecular dynamics simulations and Markov state model analysis.

    Science.gov (United States)

    Zhou, Shuangyan; Wang, Qianqian; Wang, Yuwei; Yao, Xiaojun; Han, Wei; Liu, Huanxiang

    2017-05-10

    The structural transition of prion proteins from a native α-helix (PrP C ) to a misfolded β-sheet-rich conformation (PrP Sc ) is believed to be the main cause of a number of prion diseases in humans and animals. Understanding the molecular basis of misfolding and aggregation of prion proteins will be valuable for unveiling the etiology of prion diseases. However, due to the limitation of conventional experimental techniques and the heterogeneous property of oligomers, little is known about the molecular architecture of misfolded PrP Sc and the mechanism of structural transition from PrP C to PrP Sc . The prion fragment 127-147 (PrP127-147) has been reported to be a critical region for PrP Sc formation in Gerstmann-Straussler-Scheinker (GSS) syndrome and thus has been used as a model for the study of prion aggregation. In the present study, we employ molecular dynamics (MD) simulation techniques to study the conformational change of this fragment that could be relevant to the PrP C -PrP Sc transition. Employing extensive replica exchange molecular dynamics (REMD) and conventional MD simulations, we sample a huge number of conformations of PrP127-147. Using the Markov state model (MSM), we identify the metastable conformational states of this fragment and the kinetic network of transitions between the states. The resulting MSM reveals that disordered random-coiled conformations are the dominant structures. A key metastable folded state with typical extended β-sheet structures is identified with Pro137 being located in a turn region, consistent with a previous experimental report. Conformational analysis reveals that intrapeptide hydrophobic interaction and two key residue interactions, including Arg136-His140 and Pro137-His140, contribute a lot to the formation of ordered extended β-sheet states. However, network pathway analysis from the most populated disordered state indicates that the formation of extended β-sheet states is quite slow (at the millisecond

  18. Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

    Science.gov (United States)

    Krishnan, Shuba; Paredes, João A; Zhou, Xiaoshan; Kuiper, Raoul V; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-11-21

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2(-/-)) mice extended the life span of Tk2(-/-) mice from 3 weeks to at least 20 months. The Dm-dNK(+/-)Tk2(-/-) mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK(+/-)Tk2(-/-) mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK(+/-) mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

    Science.gov (United States)

    Domino, Steven E.; Karnak, David M.; Hurd, Elizabeth A.

    2006-01-01

    Background/Aims: Neoplasia-related alterations in cell surface α(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung, and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-β signaling gene Smad3 (Madh3) were tested for α(1,2)fucosylated glycan expression. Methods: Ulex Europaeus Agglutinin-I lectin staining, fucosyltransferase gene northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed. Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express α(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of α(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3 targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knock-out mice, UEA-I lectin staining was eliminated from colon and colon tumors, however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype. Conclusions: In this model of colorectal cancer, cell surface α(1,2)fucosylation does not affect development of colon tumors. PMID:17264540

  20. Ablation of neurons expressing melanin-concentrating hormone (MCH) in adult mice improves glucose tolerance independent of MCH signaling.

    Science.gov (United States)

    Whiddon, Benjamin B; Palmiter, Richard D

    2013-01-30

    Melanin-concentrating hormone (MCH)-expressing neurons have been ascribed many roles based on studies of MCH-deficient mice. However, MCH neurons express other neurotransmitters, including GABA, nesfatin, and cocaine-amphetamine-regulated transcript. The importance of these other signaling molecules made by MCH neurons remains incompletely characterized. To determine the roles of MCH neurons in vivo, we targeted expression of the human diphtheria toxin receptor (DTR) to the gene for MCH (Pmch). Within 2 weeks of diphtheria toxin injection, heterozygous Pmch(DTR/+) mice lost 98% of their MCH neurons. These mice became lean but ate normally and were hyperactive, especially during a fast. They also responded abnormally to psychostimulants. For these phenotypes, ablation of MCH neurons recapitulated knock-out of MCH, so MCH appears to be the critical neuromodulator released by these neurons. In contrast, MCH-neuron-ablated mice showed improved glucose tolerance when compared with MCH-deficient mutant mice and wild-type mice. We conclude that MCH neurons regulate glucose tolerance through signaling molecules other than MCH.

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

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

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

  2. Muscle Glycogen Remodeling and Glycogen Phosphate Metabolism following Exhaustive Exercise of Wild Type and Laforin Knockout Mice*

    Science.gov (United States)

    Irimia, Jose M.; Tagliabracci, Vincent S.; Meyer, Catalina M.; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2015-01-01

    Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease. PMID:26216881

  3. The α-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel β-sheet structure in PrP fibrils: evidence from solid state nuclear magnetic resonance.

    Science.gov (United States)

    Tycko, Robert; Savtchenko, Regina; Ostapchenko, Valeriy G; Makarava, Natallia; Baskakov, Ilia V

    2010-11-09

    We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals cannot be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173−224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90−231 of the human PrP sequence, formed under somewhat different conditions [Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18946−18951], suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

  4. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice.

    Science.gov (United States)

    Seibenhener, Michael L; Wooten, Michael C

    2015-02-06

    Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

  5. Evidence that bank vole PrP is a universal acceptor for prions.

    Directory of Open Access Journals (Sweden)

    Joel C Watts

    2014-04-01

    Full Text Available Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP, we inoculated Tg(M109 and Tg(I109 mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109 and Tg(I109 mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD subtype MM1, into Tg(M109 mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109 mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109 mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates--including the size of proteinase K-resistant PrPSc, the pattern of cerebral PrPSc deposition, and the conformational stability--were remarkably conserved upon serial passage in Tg(M109 mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.

  6. In search of a consensus terminology in the field of platelet concentrates for surgical use: platelet-rich plasma (PRP), platelet-rich fibrin (PRF), fibrin gel polymerization and leukocytes.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Bielecki, Tomasz; Mishra, Allan; Borzini, Piero; Inchingolo, Francesco; Sammartino, Gilberto; Rasmusson, Lars; Everts, Peter A

    2012-06-01

    In the field of platelet concentrates for surgical use, most products are termed Platelet-Rich Plasma (PRP). Unfortunately, this term is very general and incomplete, leading to many confusions in the scientific database. In this article, a panel of experts discusses this issue and proposes an accurate and simple terminology system for platelet concentrates for surgical use. Four main categories of products can be easily defined, depending on their leukocyte content and fibrin architecture: Pure Platelet-Rich Plasma (P-PRP), such as cell separator PRP, Vivostat PRF or Anitua's PRGF; Leukocyteand Platelet-Rich Plasma (L-PRP), such as Curasan, Regen, Plateltex, SmartPReP, PCCS, Magellan, Angel or GPS PRP; Pure Plaletet-Rich Fibrin (P-PRF), such as Fibrinet; and Leukocyte- and Platelet-Rich Fibrin (L-PRF), such as Choukroun's PRF. P-PRP and L-PRP refer to the unactivated liquid form of these products, their activated versions being respectively named P-PRP gels and L-PRP gels. The purpose of this search for a terminology consensus is to plead for a more serious characterization of these products. Researchers have to be aware of the complex nature of these living biomaterials, in order to avoid misunderstandings and erroneous conclusions. Understanding the biomaterials or believing in the magic of growth factors ? From this choice depends the future of the field.

  7. Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication*

    Science.gov (United States)

    Salamat, Khalid; Moudjou, Mohammed; Chapuis, Jérôme; Herzog, Laetitia; Jaumain, Emilie; Béringue, Vincent; Rezaei, Human; Pastore, Annalisa; Laude, Hubert; Dron, Michel

    2012-01-01

    The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrPSc. We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrPSc and elucidate the conformational changes underlying prions generation. PMID:22511770

  8. Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication.

    Science.gov (United States)

    Salamat, Khalid; Moudjou, Mohammed; Chapuis, Jérôme; Herzog, Laetitia; Jaumain, Emilie; Béringue, Vincent; Rezaei, Human; Pastore, Annalisa; Laude, Hubert; Dron, Michel

    2012-06-01

    The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

  9. α-Synuclein Amyloids Hijack Prion Protein to Gain Cell Entry, Facilitate Cell-to-Cell Spreading and Block Prion Replication.

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    Aulić, Suzana; Masperone, Lara; Narkiewicz, Joanna; Isopi, Elisa; Bistaffa, Edoardo; Ambrosetti, Elena; Pastore, Beatrice; De Cecco, Elena; Scaini, Denis; Zago, Paola; Moda, Fabio; Tagliavini, Fabrizio; Legname, Giuseppe

    2017-08-30

    The precise molecular mechanism of how misfolded α-synuclein (α-Syn) accumulates and spreads in synucleinopathies is still unknown. Here, we show the role of the cellular prion protein (PrP C ) in mediating the uptake and the spread of recombinant α-Syn amyloids. The in vitro data revealed that the presence of PrP C fosters the higher uptake of α-Syn amyloid fibrils, which was also confirmed in vivo in wild type (Prnp +/+ ) compared to PrP knock-out (Prnp -/- ) mice. Additionally, the presence of α-Syn amyloids blocked the replication of scrapie prions (PrP Sc ) in vitro and ex vivo, indicating a link between the two proteins. Indeed, whilst PrP C is mediating the internalization of α-Syn amyloids, PrP Sc is not able to replicate in their presence. This observation has pathological relevance, since several reported case studies show that the accumulation of α-Syn amyloid deposits in Creutzfeldt-Jakob disease patients is accompanied by a longer disease course.

  10. Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

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

    2015-01-01

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

  11. Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

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    Fumiaki Yokoi

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

  12. PLATELET-RICH PLASMA (PRP AND ITS APPLICATION IN THE TREATMENT OF CHRONIC AND HARD-TO-HEAL SKIN WOUNDS. A Review.

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    Tsvetan Sokolov

    2015-12-01

    Full Text Available In the last few years various methods are being applied in the use of platelet-rich plasma (PRP during treatment in different orthopedic disease. They allow improvement of local biological condition and regeneration of different types of tissues. PRP is a modern treatment strategy with worldwide recognition. There is a high concentration of platelet growth factors in small amounts of plasma. PRP and its various forms have become one of the best methods to support the healing process of various tissues. PRP is used in regenerative medicine, because it provides two of three components (growth factors and scaffolds necessary for complete tissue regeneration. The particular reason for the appearance of lesions is important in order to select an appropriate treatment method and technical application. PRP may be used for treatment of various chronic and hard-to-heal cutaneous wounds, especially when standard conventional therapy is not good enough and surgical treatment is not possible. It reduces the duration, cost of treatment and the hospital stay. There is reduction of wound pain after starting the treatment, reduced risk of blood-borne disease transmission, wound healing is restored, and local immunity is activated.

  13. Maximal Oxygen Consumption is Reduced in Aquaporin-1 Knockout Mice

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    Samer Al-Samir

    2016-08-01

    Full Text Available We have measured maximal oxygen consumption (V’O2,max of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9 and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V’O2,max as determined by the Helox technique is reduced by ~ 16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V’O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2 by pulse oximetry. Neither under normoxic (inspiratory O2 21% nor under hypoxic conditions (11% O2 is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V’O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V’O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V’O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V’O2,max, which constitutes a new phenotype of these mice.

  14. Multiple sleep alterations in mice lacking cannabinoid type 1 receptors.

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    Alessandro Silvani

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

  15. MGE-derived nNOS+ interneurons promote fear acquisition in nNOS-/- mice.

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    Zhang, Lin; Yuan, Hong-Jin; Cao, Bo; Kong, Cheng-Cheng; Yuan, Fang; Li, Jun; Ni, Huan-Yu; Wu, Hai-Yin; Chang, Lei; Liu, Yan; Luo, Chun-Xia

    2017-12-02

    Neuronal nitric oxide synthase (nNOS) 1 , mainly responsible for NO release in central nervous system (CNS) 2 , plays a significant role in multiple physiological functions. However, the function of nNOS + interneurons in fear learning has not been much explored. Here we focused on the medial ganglionic eminences (MGE) 3 -derived nNOS + interneurons in fear learning. To determine the origin of nNOS + interneurons, we cultured neurons in vitro from MGE, cortex, lateral ganglionic eminence (LGE) 4 , caudal ganglionic eminences (CGE) 5 and preoptic area (POA) 6 . The results showed that MGE contained the most abundant precursors of nNOS + interneurons. Moreover, donor cells from E12.5 embryos demonstrated the highest positive rate of nNOS + interneurons compared with other embryonic periods (E11.5, E12, E13, E13.5 and E14). Additionally, these cells from E12.5 embryos showed long axonal and abundant dendritic arbors after 10 days culture, indicating the capability to disperse and integrate in host neural circuits after transplantation. To investigate the role of MGE-derived nNOS + interneurons in fear learning, donor MGE cells were transplanted into dentate gyrus (DG) 7 of nNOS knock-out (nNOS -/- ) or wild-type mice. Results showed that the transplantation of MGE cells promoted the acquisition of nNOS -/- but not the wild-type mice, suggesting the importance of nNOS + neurons in fear acquisition. Moreover, we transplanted MGE cells from nNOS -/- mice or wild-type mice into DG of the nNOS -/- mice and found that only MGE cells from wild-type mice but not the nNOS -/- mice rescued the deficit in acquisition of the nNOS -/- mice, further confirming the positive role of nNOS + neurons in fear learning. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice.

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    Antonino Sgroi

    Full Text Available BACKGROUND: Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration. METHODS: We performed 70%-hepatectomy in wild type (WT mice, IL-1ra knock-out (KO mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU incorporation, proliferating cell nuclear antigen (PCNA and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes. RESULTS: At 24h and at 48 h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1 and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment. CONCLUSION: IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.

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

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

    2018-03-01

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

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

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

    2016-06-30

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

  19. Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine attenuates liver fibrogenesis in mice.

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    Catalina Atorrasagasti

    Full Text Available INTRODUCTION: Secreted Protein, Acidic and Rich in Cysteine (SPARC is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. METHODS: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+ and knock-out (SPARC(-/- mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/- and SPARC(+/+ mice using Affymetrix Mouse Gene ST 1.0 array. RESULTS: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/- mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/- mice when compared to SPARC(+/+ mice; in addition, MMP-2 expression was increased in SPARC(-/- mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. CONCLUSIONS: Overall our data suggest that

  20. Earlier onset of motor deficits in mice with double mutations in Dyt1 and Sgce.

    Science.gov (United States)

    Yokoi, Fumiaki; Yang, Guang; Li, Jindong; DeAndrade, Mark P; Zhou, Tong; Li, Yuqing

    2010-10-01

    DYT1 early-onset generalized torsion dystonia is an inherited movement disorder caused by mutations in DYT1 coding for torsinA with ∼30% penetrance. Most of the DYT1 dystonia patients exhibit symptoms during childhood and adolescence. On the other hand, DYT1 mutation carriers without symptoms during these periods mostly do not exhibit symptoms later in their life. Little is known about what controls the timing of the onset, a critical issue for DYT1 mutation carriers. DYT11 myoclonus-dystonia is caused by mutations in SGCE coding for ε-sarcoglycan. Two dystonia patients from a single family with double mutations in DYT1 and SGCE exhibited more severe symptoms. A recent study suggested that torsinA contributes to the quality control of ε-sarcoglycan. Here, we derived mice carrying mutations in both Dyt1 and Sgce and found that these double mutant mice showed earlier onset of motor deficits in beam-walking test. A novel monoclonal antibody against mouse ε-sarcoglycan was developed by using Sgce knock-out mice to avoid the immune tolerance. Western blot analysis suggested that functional deficits of torsinA and ε-sarcoglycan may independently cause motor deficits. Examining additional mutations in other dystonia genes may be beneficial to predict the onset in DYT1 mutation carriers.

  1. Dampened dopamine-mediated neuromodulation in prefrontal cortex of fragile X mice.

    Science.gov (United States)

    Paul, Kush; Venkitaramani, Deepa V; Cox, Charles L

    2013-02-15

    Fragile X syndrome (FXS) is the most common form of inheritable mental retardation caused by transcriptional silencing of the Fmr1 gene resulting in the absence of fragile X mental retardation protein (FMRP). The role of this protein in neurons is complex and its absence gives rise to diverse alterations in neuronal function leading to neurological disorders including mental retardation, hyperactivity, cognitive impairment, obsessive-compulsive behaviour, seizure activity and autism. FMRP regulates mRNA translation at dendritic spines where synapses are formed, and thus the lack of FMRP can lead to disruptions in synaptic transmission and plasticity. Many of these neurological deficits in FXS probably involve the prefrontal cortex, and in this study, we have focused on modulatory actions of dopamine in the medial prefrontal cortex. Our data indicate that dopamine produces a long-lasting enhancement of evoked inhibitory postsynaptic currents (IPSCs) mediated by D1-type receptors seen in wild-type mice; however, such enhancement is absent in the Fmr1 knock-out (Fmr1 KO) mice. The facilitation of IPSCs produced by direct cAMP stimulation was unaffected in Fmr1 KO, but D1 receptor levels were reduced in these animals. Our results show significant disruption of dopaminergic modulation of synaptic transmission in the Fmr1 KO mice and this alteration in inhibitory activity may provide insight into potential targets for the rescue of deficits associated with FXS.

  2. Estrogen-related receptor gamma and hearing function: evidence of a role in humans and mice.

    Science.gov (United States)

    Nolan, Lisa S; Maier, Hannes; Hermans-Borgmeyer, Irm; Girotto, Giorgia; Ecob, Russell; Pirastu, Nicola; Cadge, Barbara A; Hübner, Christian; Gasparini, Paolo; Strachan, David P; Davis, Adrian; Dawson, Sally J

    2013-08-01

    Since estrogen is thought to protect pre-menopausal women from age-related hearing loss, we investigated whether variation in estrogen-signalling genes is linked to hearing status in the 1958 British Birth Cohort. This analysis implicated the estrogen-related receptor gamma (ESRRG) gene in determining adult hearing function and was investigated further in a total of 6134 individuals in 3 independent cohorts: (i) the 1958 British Birth Cohort; (ii) a London ARHL case-control cohort; and (iii) a cohort from isolated populations of Italy and Silk Road countries. Evidence of an association between the minor allele of single nucleotide polymorphism (SNP) rs2818964 and hearing status was found in females, but not in males in 2 of these cohorts: p = 0.0058 (London ARHL) and p = 0.0065 (Carlantino, Italy). Furthermore, assessment of hearing in Esrrg knock-out mice revealed a mild 25-dB hearing loss at 5 weeks of age. At 12 weeks, average hearing thresholds in female mice((-/-)) were 15 dB worse than in males((-/-)). Together these data indicate ESRRG plays a role in maintenance of hearing in both humans and mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

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    Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C

    2015-09-01

    Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets

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    Alquier, Thierry; Peyot, Marie-Line; Latour, M. G.; Kebede, Melkam; Sorensen, Christina M.; Gesta, Stephane; Kahn, C. R.; Smith, Richard D.; Jetton, Thomas L.; Metz, Thomas O.; Prentki, Marc; Poitout, Vincent J.

    2009-11-01

    The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

  5. Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice.

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    Caradu, Caroline; Guy, Alexandre; James, Chloé; Reynaud, Annabel; Gadeau, Alain-Pierre; Renault, Marie-Ange

    2018-04-01

    Hedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated. The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis. To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes. Although ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.

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

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

    2018-06-01

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

  7. Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b

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    Wenteng He

    2018-02-01

    Full Text Available Summary: Androgenetic haploid embryonic stem cells (AG-haESCs hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood. : Ectopic expression of Dnmt3b could rescue DNA methylation level in repetitive sequences of hypomethylated AG-haESCs, suppress high incidence of self-diploidization, and promote developmental potential of SC embryos, and most SC mice could survive into adulthood. Keywords: androgenetic haploid embryonic stem cells, self-diploidization, semi-cloned mice, DNA methylation, Dnmt3b

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

    Directory of Open Access Journals (Sweden)

    Sun Wei-Zen

    2008-09-01

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

  9. The Metalloproteinase ADAM28 Promotes Metabolic Dysfunction in Mice

    Directory of Open Access Journals (Sweden)

    Lakshini Herat

    2017-04-01

    Full Text Available Obesity and diabetes are major causes of morbidity and mortality globally. The current study builds upon our previous association studies highlighting that A Disintegrin And Metalloproteinase 28 (ADAM28 appears to be implicated in the pathogenesis of obesity and type 2 diabetes in humans. Our novel study characterised the expression of ADAM28 in mice with the metabolic syndrome and used molecular inhibition approaches to investigate the functional role of ADAM28 in the pathogenesis of high fat diet-induced obesity. We identified that ADAM28 mRNA and protein expression was markedly increased in the livers of mice with the metabolic syndrome. In addition, noradrenaline, the major neurotransmitter of the sympathetic nervous system, results in elevated Adam28 mRNA expression in human monocytes. Downregulation of ADAM28 with siRNA technology resulted in a lack of weight gain, promotion of insulin sensitivity/glucose tolerance and decreased liver tumour necrosis factor-α (TNF-α levels in our diet-induced obesity mouse model as well as reduced blood urea nitrogen, alkaline phosphatase and aspartate aminotransferase. In addition, we show that ADAM28 knock-out mice also displayed reduced body weight, elevated high density lipoprotein cholesterol levels, and reductions in blood urea nitrogen, alkaline phosphatase, and aspartate aminotransferase. The results of this study provide important insights into the pathogenic role of the metalloproteinase ADAM28 in the metabolic syndrome and suggests that downregulation of ADAM28 may be a potential therapeutic strategy in the metabolic syndrome.

  10. Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice

    Directory of Open Access Journals (Sweden)

    Mühlfeld Christian

    2007-10-01

    Full Text Available Abstract Background Surfactant protein D (SP-D deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. Methods SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. Main Results After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. Conclusion Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are

  11. Postnatal Ablation of Synaptic Retinoic Acid Signaling Impairs Cortical Information Processing and Sensory Discrimination in Mice.

    Science.gov (United States)

    Park, Esther; Tjia, Michelle; Zuo, Yi; Chen, Lu

    2018-06-06

    Retinoic acid (RA) and its receptors (RARs) are well established essential transcriptional regulators during embryonic development. Recent findings in cultured neurons identified an independent and critical post-transcriptional role of RA and RARα in the homeostatic regulation of excitatory and inhibitory synaptic transmission in mature neurons. However, the functional relevance of synaptic RA signaling in vivo has not been established. Here, using somatosensory cortex as a model system and the RARα conditional knock-out mouse as a tool, we applied multiple genetic manipulations to delete RARα postnatally in specific populations of cortical neurons, and asked whether synaptic RA signaling observed in cultured neurons is involved in cortical information processing in vivo Indeed, conditional ablation of RARα in mice via a CaMKIIα-Cre or a layer 5-Cre driver line or via somatosensory cortex-specific viral expression of Cre-recombinase impaired whisker-dependent texture discrimination, suggesting a critical requirement of RARα expression in L5 pyramidal neurons of somatosensory cortex for normal tactile sensory processing. Transcranial two-photon imaging revealed a significant increase in dendritic spine elimination on apical dendrites of somatosensory cortical layer 5 pyramidal neurons in these mice. Interestingly, the enhancement of spine elimination is whisker experience-dependent as whisker trimming rescued the spine elimination phenotype. Additionally, experiencing an enriched environment improved texture discrimination in RARα-deficient mice and reduced excessive spine pruning. Thus, RA signaling is essential for normal experience-dependent cortical circuit remodeling and sensory processing. SIGNIFICANCE STATEMENT The importance of synaptic RA signaling has been demonstrated in in vitro studies. However, whether RA signaling mediated by RARα contributes to neural circuit functions in vivo remains largely unknown. In this study, using a RARα conditional

  12. Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

    Science.gov (United States)

    Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

    2018-01-01

    Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

  13. Smoothing of the Time Structure of Slowly Extracted Beam From Synchrotron by RF-Knock-out Method

    International Nuclear Information System (INIS)

    Voloshnyuk, A.V.; Bezshyjko, O.A.; Dolinskiy, A.V.; Dolinskij, A.V.

    2005-01-01

    Results of the study are presented in work on smoothing of the time structure of the bunch, slowly extracted from synchrotron. The numerical algorithm has been designed for study of the influence of the radio-frequency field of the resonator on time structure of the bunch. The numerical algorithm is based on method Monte-Carlo, where particles in the beam have been extracted by means of slow moving to the third-order resonance conditions. Characteristics of the time structure are vastly smoothed when synchrotron oscillations have been used as first experiments showed. Theoretical motivation of the reasons, influencing upon time structure of the slowly extracted beam is explained in given work

  14. CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection.

    Science.gov (United States)

    Sessions, John W; Skousen, Craig S; Price, Kevin D; Hanks, Brad W; Hope, Sandra; Alder, Jonathan K; Jensen, Brian D

    2016-01-01

    CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times). Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples. These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.

  15. Neutrino-nucleus cross-sections: a unified theoretical approach for nucleon knock-out, coherent and incoherent pion production

    CERN Document Server

    Martini, M; G. Chanfray; Marteau, J

    2010-01-01

    Neutrino-nucleus cross-sections are needed to interpret neutrino oscillation data, as neutrino detectors involve complex nuclei. We present a theory of neutrino interactions with nuclei aimed at a unified description of the partial cross-sections, namely quasi-elastic and multi-nucleon emission, coherent and incoherent single pion production. We compare our approach to the available neutrino experimental data on carbon. We also discuss the evolution of the neutrino cross-sections with the mass number in view of future precision ex- periments which will use a liquid argon chamber.

  16. A Unified approach for nucleon knock-out, coherent and incoherent pion production in neutrino interactions with nuclei

    CERN Document Server

    Martini, M.; Chanfray, G.; Marteau, J.

    2009-01-01

    We present a theory of neutrino interactions with nuclei aimed at the description of the partial cross-sections, namely quasi-elastic and multi-nucleon emission, coherent and incoherent single pion production. For this purpose, we use the theory of nuclear responses treated in the random phase approximation, which allows a unified description of these channels. It is particularly suited for the coherent pion production where collective effects are important whereas they are moderate in the other channels. We also study the evolution of the neutrino cross-sections with the mass number from carbon to calcium. We compare our approach to the available neutrino experimental data on carbon. We put a particular emphasis on the multi-nucleon channel, which at present is not easily distinguishable from the quasi-elastic events. This component turns out to be quite relevant for the interpretation of experiments (K2K, MiniBooNE, SciBooNE). It can account in particular for the unexpected behavior of the quasi-elastic cro...

  17. Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-Out System

    National Research Council Canada - National Science Library

    Czarneski, Jennifer

    2000-01-01

    .... The hypothesis of the project was to develop a novel breast cancer model using the tissue-specific expression of the Mtv-17 locus, which was previously shown by this lab to only be expressed in the mammary gland...

  18. Sun-Earth Connections: How the Sun Knocks Out My Cell Phone from 150 Million Kilometers Away

    Science.gov (United States)

    Ladbury, Raymond L.

    2014-01-01

    Large solar particle events (SPE) threaten many elements of critical infrastructure. A 2013 study by Lloyds of London and Atmospheric and Environmental Research recently found that if a worst-case solar event like the 1859 Carrington Event struck our planet now, it could result on $0.6-$2.36 trillion in damages to the economy. In March 2014, researchers Y. D. Liu et al. revealed that just such an event had narrowly missed Earth in July 2012. The event was observed by the STEREO A spacecraft. In this presentation, we examine how the sun can pack such a punch from 150 million km away, the threats such solar particle events pose, their mechanisms and the efforts NASA and other space agencies are carrying out to understand and mitigate such risks.

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

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, V.; Rülicke, T.; Rathkolb, B.; Hans, W.; Bohla, A.; Eickelberg, O.; Stoeger, T.; Wolf, E.; Yildirim, A.Ö.; Gailus-Durner, V.; Fuchs, H.; de Angelis, M.H.; Hozák, Pavel

    2013-01-01

    Roč. 8, č. 4 (2013), e61406 E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020022; GA MŠk LH12143; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : nuclear myosin * myosin isoforms * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  20. Evaluation of the effect of platelet rich plasma (PRP) on enhancement of bone healing in diaphyseal bone defects by radiography and computed tomography

    International Nuclear Information System (INIS)

    Özak, Ahmet; Yardimci, Cenk; Nİsbet, Özlem H.; Bayrak, İlkay Koray; Nİsbet, Cevat

    2010-01-01

    The effect of platelet-rich plasma (PRP) with autogenous cancellous bone graft on enhancement of bone healing in diaphyseal bone defects was evaluated. A 4-mm defect was created in the middiaphysis of the tibias of 20 rabbits. Rabbits were divided into two groups of ten animals each: only autogenous cancellous graft, PRP and autogenous cancellous graft. In animals of group 1, only autogenous cancellous grafts, and to those in group 2, PRP and autogenous cancellous grafts, were applied to the defect. Radiographical and computed tomography (CT) views were taken and evaluated on postoperative days 0, 15, 30, 60, and 90. According to the bone formation, union, and remodeling scores, group 1 had better scores than group 2 on days 30, 60, and 90. The density was significantly increased on day 60 than on days 0, 15, and 30 in group 1. In conclusion, it was evaluated that PRP could not enhance the bone regeneration in diaphyseal defects when used with autogenous cancellous bone graft

  1. Impact of strong selection for the PrP major gene on genetic variability of four French sheep breeds (Open Access publication

    Directory of Open Access Journals (Sweden)

    Pantano Thais

    2008-11-01

    Full Text Available Abstract Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR "resistant" allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3–4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers. The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.

  2. Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro

    DEFF Research Database (Denmark)

    Bergstrøm, Linda Alice; Hvass, Henriette Cordes; Zsurger, N.

    2005-01-01

    One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrPSc) of the cellular prion protein (PrPC) primarily in the central nervous system. The synthetic prion peptide PrP106-126 shares many characteristics...

  3. Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of the human spliceosomal DExD/H-box protein hPrp22

    International Nuclear Information System (INIS)

    Kudlinzki, Denis; Nagel, Christian; Ficner, Ralf

    2009-01-01

    The cloning, purification and crystallization of the C-terminal domain of human hPrp22 are reported. This communication also contains data for the preliminary X-ray diffraction analysis. The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950–1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector. The protein was subsequently overproduced, purified and crystallized. The crystals obtained diffracted to 2.1 Å resolution, belonged to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = b = 78.2, c = 88.4 Å, and contained one molecule in the asymmetric unit

  4. Robust nonlinear PID-like fuzzy logic control of a planar parallel (2PRP-PPR) manipulator.

    Science.gov (United States)

    Londhe, P S; Singh, Yogesh; Santhakumar, M; Patre, B M; Waghmare, L M

    2016-07-01

    In this paper, a robust nonlinear proportional-integral-derivative (PID)-like fuzzy control scheme is presented and applied to complex trajectory tracking control of a 2PRP-PPR (P-prismatic, R-revolute) planar parallel manipulator (motion platform) with three degrees-of-freedom (DOF) in the presence of parameter uncertainties and external disturbances. The proposed control law consists of mainly two parts: first part uses a feed forward term to enhance the control activity and estimated perturbed term to compensate for the unknown effects namely external disturbances and unmodeled dynamics, and the second part uses a PID-like fuzzy logic control as a feedback portion to enhance the overall closed-loop stability of the system. Experimental results are presented to show the effectiveness of the proposed control scheme. Copyright © 2016 ISA. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-01-15

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

  6. Deficiency of C5L2 increases macrophage infiltration and alters adipose tissue function in mice.

    Directory of Open Access Journals (Sweden)

    Danny Gauvreau

    Full Text Available BACKGROUND: Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP, and C5a, involved in innate immunity. AIM: We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl and C5L2 knock-out (C5L2(-/- mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO conditions. RESULTS: In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold both over time and with DIO. By contrast, in C5L2(-/-, there was no change in C5aR in WAT. C5L2(-/- mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro- vs M2 (anti-inflammatory macrophage proportion was unchanged but C5L2(-/- adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2(-/- mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2(-/- mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance. CONCLUSION: Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.

  7. Is Platelet-Rich Plasma (PRP) Effective in the Treatment of Acute Muscle Injuries? A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Grassi, Alberto; Napoli, Francesca; Romandini, Iacopo; Samuelsson, Kristian; Zaffagnini, Stefano; Candrian, Christian; Filardo, Giuseppe

    2018-04-01

    Muscle lesions account for one-third of sport-related injuries, thus representing a substantial problem for both players and their teams. The use of platelet-rich plasma (PRP) injections is rapidly growing in clinical practice, prompted by an unmet clinical need with a large commercial market. However, after early reports of positive preliminary experience, higher quality studies recently questioned the real benefit provided by PRP injections to promote muscle healing and return to sport. To evaluate the effect of platelet-rich plasma (PRP) injections on outcomes following acute muscle injuries. Meta-analysis of randomized, controlled trials (RCTs), Level I. PubMed (MEDLINE), Cochrane (CENTRAL), Web of Science, clinicaltrials.gov, who.int, isrctn.com, greylit.org, opengrey.eu. RCTs investigating the effect of PRP for the treatment of acute muscle injuries against at least one control group including patients treated with placebo injection or physical therapy. The outcomes evaluated were time to return to sport, re-injuries, complications, pain, muscle strength, range of motion (ROM)/flexibility, muscle function, and imaging. Six studies, involving 374 patients, were included in the meta-analysis. The time to return to sport evaluated in all six studies was significantly shorter in patients treated with PRP (mean difference = - 7.17 days). However, if only the double-blind studies (n = 2) or studies including only hamstring injuries (n = 3) were considered, non-significant differences were found. Re-injuries (relative risk = - 0.03) and complications (relative risk = 0.01) were also similar between the two groups (p > 0.05), nor were any substantial differences found regarding pain, muscle strength, ROM/flexibility, muscle function, and imaging. The performance bias was high risk due to the lack of patient blinding in four studies. The quality of evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) was

  8. PrP expression, PrPSc accumulation and innervation of splenic compartments in sheep experimentally infected with scrapie.

    Directory of Open Access Journals (Sweden)

    Randi Sørby

    Full Text Available BACKGROUND: In prion disease, the peripheral expression of PrP(C is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP(Sc accumulation, localisation of nerve fibres and PrP(C expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep. METHODOLOGY/PRINCIPAL FINDINGS: Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP(C and PrP(Sc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP(Sc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP(Sc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP(Sc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP(Sc-laden germinal centres. However, the close association between nerves and PrP(Sc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres. CONCLUSIONS/SIGNIFICANCE: The findings suggest that the degree of PrP(Sc accumulation does not depend on the expression level of PrP(C. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP(Sc.

  9. Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

    Science.gov (United States)

    Wang, Xin; Shi, Qi; Xu, Kun; Gao, Chen; Chen, Cao; Li, Xiao-Li; Wang, Gui-Rong; Tian, Chan; Han, Jun; Dong, Xiao-Ping

    2011-01-01

    Background Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. Methodology/Principal Findings To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. Conclusions/Significance The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants

  10. Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Xin Wang

    Full Text Available BACKGROUND: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C to the pathogenic one (PrP(Sc. The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. METHODOLOGY/PRINCIPAL FINDINGS: To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER, the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL and PrP with three amino acids exchange in transmembrane region (PrP-3AV were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K failed. CONCLUSIONS/SIGNIFICANCE: The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them

  11. Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs.

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    Beshkar, Pezhman; Hosseini, Ehteramolsadat; Ghasemzadeh, Mehran

    2018-02-01

    Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study. PRP- and BC-platelet concentrates (PCs) were subjected to flowcytometry analysis to examine the expression of platelet activation marker, P-selectin as well as active confirmation of the GPIIb/IIIa (α IIb β 3 ) on day 0, 1, 3 and 5 post-storage. Platelet adhesion to fibrinogen matrix was evaluated by fluorescence microscopy. Glucose concentration and LDH activity were also measured by colorimetric methods. The increasing P-selectin expression during storage was in a reverse correlation with PAC-1 binding (r = -0.67; p = .001). PRP-PCs showed the higher level of P-selectin expression than BC-PCs, whereas the levels of PAC-1 binding and platelet adhesion to fibrinogen matrix were significantly lower in PRP-PCs. Higher levels of active confirmation of the GPIIb/IIIa in BC-PCs were also associated with greater concentration of glucose in these products. We demonstrated the superior capacities of integrin activation and adhesion to fibrinogen for BC-PCs compared to those of PRP-PCs. These findings may provide more advantages for BC method of platelet preparation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Functional assessment of autologous platelet-rich plasma (PRP) after long-term storage at -20 °C without any preservation agent.

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    Hosnuter, Mubin; Aslan, Cem; Isik, Daghan; Caliskan, Gorkem; Arslan, Banu; Durgun, Mustafa

    2017-08-01

    Platelet-rich plasma (PRP) is increasingly being used in the treatment of chronic wounds, pathologies of the musculoskeletal system, and in cosmetic medicine; however, the preparation of platelet-rich plasma is both time-consuming and requires invasive intervention. Additional costs are introduced if special equipment is used during preparation. The aim of the present study is to test whether autologous platelet-rich plasma (PRP) preserves the feature of growth factor release when stored at -20 °C after preparation. Autologous PRP concentrates were prepared using whole blood samples obtained from 20 healthy subjects and divided into three parts to form three groups. Epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet derived growth factor-AB (PDGF-AB), insulin-like growth factor 1 (IGF-1), transforming growth factor-beta (TGF-β), and P-Selectin levels were immediately analysed in the control group. The other groups were defined as the experimental groups and were stored at -20 °C and analysed on the 7th and the 14th days. The same growth factors were tested in the experimental groups. The growth factors (EGF, VEGF, PDGF-AB, IGF-1, TGF-β) and P-selectin levels were significantly decreased in the autologous PRP samples stored at -20 °C compared to the control group. The growth factor levels on days 7 and 14 suggest that autologous PRP can be stored at -20 °C without preservative agents, although in vivo studies are required in order to evaluate the clinical efficacy of the detected growth factor levels.

  13. Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.

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    Arora, Satyam; Doda, Veena; Kotwal, Urvershi; Dogra, Mitu

    2016-02-01

    Platelet derived biomaterials represent a key source of cytokines and growth factors extensively used for tissue regeneration; wound healing and tissue repair. Our study was to quantify platelets and growth factors released by PRP when prepared at different centrifugal force (g) and time. Our study was approved by the institutional ethical committee. One hundred millilitres of whole blood (WB) was collected in bag with CPDA as the anticoagulant(AC); (14 mL for 100 mL WB ratio). Nine aliquots of 10 mL each were made from the bag and set of three aliquots were made a group. PRP was prepared at varying centrifugal force (group A: -110 g, group B: -208 g & group C: -440 g) & time (1: -5 min, 2: -10 min & 3: -20 min). Contents of each PRP prepared were analysed. Commercial sandwich ELISA kits were used to quantify the concentrations of CD62P (Diaclone SAS; France), Platelet derived growth factors-AB (Qayee-Bio; China), transforming growth factor-β1 (DRG; Germany) and vascular endothelial growth factor (Boster Immuno Leader; USA) released in each PRP prepared. Eight volunteers were enrolled in the study (24-30 years). The baseline blood counts of all the volunteers were comparable (p ≥ 0.05). Mean ± SD of platelet yield of all nine groups ranged from 17.2 ± 4.2% to 78.7 ± 5.7%. Each PRP was activated with calcified thromboplastin to quantify the growth factors released by them. Significantly higher (p < 0.05) transforming growth factor-β1 and vascular endothelial growth factor were released compared to the baseline. Our study highlights the variation in both force (g) and time results in changes at cellular level and growth factor concentrations. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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    Morgan Kristen

    2011-01-01

    Full Text Available Abstract 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

  15. Characterization of Variant Creutzfeldt-Jakob Disease Prions in Prion Protein-humanized Mice Carrying Distinct Codon 129 Genotypes*

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    Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W.; Mohri, Shirou; Kitamoto, Tetsuyuki

    2013-01-01

    To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype. PMID:23792955

  16. Characterization of variant Creutzfeldt-Jakob disease prions in prion protein-humanized mice carrying distinct codon 129 genotypes.

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    Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W; Mohri, Shirou; Kitamoto, Tetsuyuki

    2013-07-26

    To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.

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

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    Toru eNakamura

    2014-07-01

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

  18. Development of a Murine Model for Aerosolized Ebolavirus Infection Using a Panel of Recombinant Inbred Mice

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    Malak Kotb

    2012-12-01

    Full Text Available Countering aerosolized filovirus infection is a major priority of biodefense research.  Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported.  A murine model of aerosolized filovirus infection in mice should be useful for screening vaccine candidates and therapies.  In this study, various strains of wild-type and immunocompromised mice were exposed to aerosolized wild-type (WT or mouse-adapted (MA Ebola virus (EBOV.  Upon exposure to aerosolized WT-EBOV, BALB/c, C57BL/6 (B6, and DBA/2 (D2 mice were unaffected, but 100% of severe combined immunodeficiency (SCID and 90% of signal transducers and activators of transcription (Stat1 knock-out (KO mice became moribund between 7–9 days post-exposure (dpe.  Exposure to MA-EBOV caused 15% body weight loss in BALB/c, but all mice recovered.  In contrast, 10–30% lethality was observed in B6 and D2 mice exposed to aerosolized MA-EBOV, and 100% of SCID, Stat1 KO, interferon (IFN-γ KO and Perforin KO mice became moribund between 7–14 dpe. In order to identify wild-type, inbred, mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 60 BXD (C57BL/6 crossed with DBA/2 recombinant inbred (RI and advanced RI (ARI mouse strains were exposed to aerosolized MA-EBOV, and monitored for disease severity. A complete spectrum of disease severity was observed. All BXD strains lost weight but many recovered. However, infection was uniformly lethal within 7 to 12 days post-exposure in five BXD strains.  Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 90–100% lethality in two strains.  Analysis of post-mortem tissue from BXD strains that became moribund and were euthanized at the lower dose of MA-EBOV, showed liver damage in all mice as well as lung lesions in

  19. Chronic minocycline treatment improves social recognition memory in adult male Fmr1 knockout mice.

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    Yau, Suk Yu; Chiu, Christine; Vetrici, Mariana; Christie, Brian R

    2016-10-01

    Fragile X syndrome (FXS) is caused by a mutation in the Fmr1 gene that leads to silencing of the gene and a loss of its gene product, Fragile X mental retardation protein (FMRP). Some of the key behavioral phenotypes for FXS include abnormal social anxiety and sociability. Here we show that Fmr1 knock-out (KO) mice exhibit impaired social recognition when presented with a novel mouse, and they display normal social interactions in other sociability tests. Administering minocycline to Fmr1 KO mice throughout critical stages of neural development improved social recognition memory in the novel mouse recognition task. To determine if synaptic changes in the prefrontal cortex (PFC) could have played a role in this improvement, we examined PSD-95, a member of the membrane-associated guanylate kinase family, and signaling molecules (ERK1/2, and Akt) linked to synaptic plasticity in the PFC. Our analyses indicated that while minocycline treatment can enhance behavioral performance, it does not enhance expression of PSD-95, ERK1/2 or Akt in the PFC. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

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    Sassoli, Chiara; Vallone, Larissa; Tani, Alessia; Chellini, Flaminia; Nosi, Daniele; Zecchi-Orlandini, Sandra

    2018-02-05

    Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

  1. Urotensin II Induces ER Stress and EMT and Increase Extracellular Matrix Production in Renal Tubular Epithelial Cell in Early Diabetic Mice

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    Xin-Xin Pang

    2016-07-01

    Full Text Available Background/Aims: Urotensin II (UII and its receptor are highly expressed in the kidney tissue of patients with diabetic nephropathy (DN. The aim of this study is to examine the roles of UII in the induction of endoplasmic reticulum stress (ER stress and Epithelial-mesenchymal transition (EMT in DN in vivo and in vitro. Methods: Kidney tissues were collected from patients with DN. C57BL/6 mice and mice with UII receptor knock out were injected with two consecutive doses of streptozotocin to induce diabetes and were sacrificed at 3th week for in vivo study. HK-2 cells in vitro were cultured and treated with UII. Markers of ER stress and EMT, fibronectin and type IV collagen were detected by immunohistochemistry, real time PCR and western blot. Results: We found that the expressions of protein of UII, GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were upregulated while E-cadherin protein was downregulated as shown by immunohistochemistry or western blot analysis in kidney of diabetic mice in comparison to normal control; moreover expressions of GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were inhibited while E-caherin expression was enhanced in kidney in diabetic mice with UII receptor knock out in comparison to C57BL/6 diabetic mice. In HK-2 cells, UII induced upregulation of GRP78, CHOP, ALPHA-SMA, fibroblast-specifc protein 1(FSP-1, fibronectin and type collagen and downregulation of E-cadherin. UII receptor antagonist can block UII-induced ER stress and EMT; moreover, 4-PBA can inhibit the mRNA expression of ALPHA-SMA and FSP1 induced by UII in HK-2 cells. Conclusions: We are the first to verify UII induces ER stress and EMT and increase extracellular matrix production in renal tubular epithelial cell in early diabetic mice. Moreover, UII may induce renal tubular epithelial EMT via triggering ER stress pathway in vitro, which might be the new pathogenic pathway for the development of renal fibrosis in DN.

  2. Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.

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    Yamaguchi, Yoshitaka; Miyata, Hironori; Uchiyama, Keiji; Ootsuyama, Akira; Inubushi, Sachiko; Mori, Tsuyoshi; Muramatsu, Naomi; Katamine, Shigeru; Sakaguchi, Suehiro

    2012-01-01

    Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.

  3. Tumor-extrinsic discoidin domain receptor 1 promotes mammary tumor growth by regulating adipose stromal interleukin 6 production in mice.

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    Sun, Xiujie; Gupta, Kshama; Wu, Bogang; Zhang, Deyi; Yuan, Bin; Zhang, Xiaowen; Chiang, Huai-Chin; Zhang, Chi; Curiel, Tyler J; Bendeck, Michelle P; Hursting, Stephen; Hu, Yanfen; Li, Rong

    2018-02-23

    Discoidin domain receptor 1 (DDR1) is a collagen receptor that mediates cell communication with the extracellular matrix (ECM). Aberrant expression and activity of DDR1 in tumor cells are known to promote tumor growth. Although elevated DDR1 levels in the stroma of breast tumors are associated with poor patient outcome, a causal role for tumor-extrinsic DDR1 in cancer promotion remains unclear. Here we report that murine mammary tumor cells transplanted to syngeneic recipient mice in which Ddr1 has been knocked out (KO) grow less robustly than in WT mice. We also found that the tumor-associated stroma in Ddr1- KO mice exhibits reduced collagen deposition compared with the WT controls, supporting a role for stromal DDR1 in ECM remodeling of the tumor microenvironment. Furthermore, the stromal-vascular fraction (SVF) of Ddr1 knockout adipose tissue, which contains committed adipose stem/progenitor cells and preadipocytes, was impaired in its ability to stimulate tumor cell migration and invasion. Cytokine array-based screening identified interleukin 6 (IL-6) as a cytokine secreted by the SVF in a DDR1-dependent manner. SVF-produced IL-6 is important for SVF-stimulated tumor cell invasion in vitro , and, using antibody-based neutralization, we show that tumor promotion by IL-6 in vivo requires DDR1. In conclusion, our work demonstrates a previously unrecognized function of DDR1 in promoting tumor growth. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.