DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

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Nielsen, Line; Jensen, Trine Hammer; Kristensen, Birte; Jensen, Tove Dannemann; Karlskov-Mortensen, Peter; Lund, Morten; Aasted, Bent; Blixenkrone-Møller, Merete

2012-10-01

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

The NLR Protein NLRP6 Does Not Impact Gut Microbiota Composition

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Paul Lemire

2017-12-01

Full Text Available NLRP6 is a Nod-like receptor expressed in the intestinal epithelium. Previous studies reported a protective role for NLRP6 against intestinal injury and colitis-associated carcinogenesis via the regulation and establishment of a healthy microbiota. However, these results were not obtained using littermate animals, leaving the possibility that the pro-colitogenic microbiota phenotype associated with knockout (KO mice was stochastically acquired and genotype independent. Here, we analyzed the microbiota at three intestinal locations from Nlrp6−/− and wild-type (WT littermates, either co-caged or individually caged after weaning. Our results demonstrate that NLRP6 does not significantly influence the intestinal microbiota at homeostasis, and they support a previously reported sex-biased microbial community structure. Moreover, WT and Nlrp6−/− littermate mice displayed comparable sensitivity to dextran sulfate sodium (DSS-induced colitis, although increased sensitivity was noted in KO females. Our results clarify the role of NLRP6 in microbiota and colitis control, and they highlight the importance of analyzing littermate animals in such studies.

Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea.

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Deepti Nair

Full Text Available In rodents, exposure to intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox(_/Y and wild-type littermates. On a standard place training task, gp91phox(_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA controls, while no changes emerged in gp91phox(_/Y mice. Additionally, wild-type mice, but not gp91phox(_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.

Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents.

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Nicaise, Charles; Prozzi, Deborah; Viaene, Eric; Moreno, Christophe; Gustot, Thierry; Quertinmont, Eric; Demetter, Pieter; Suain, Valérie; Goffin, Philippe; Devière, Jacques; Hols, Pascal

2008-10-01

Hyperammonemia is a common complication of acute and chronic liver diseases. Often accompanied with side effects, therapeutic interventions such as antibiotics or lactulose are generally targeted to decrease the intestinal production and absorption of ammonia. In this study, we aimed to modulate hyperammonemia in three rodent models by administration of wild-type Lactobacillus plantarum, a genetically engineered ammonia hyperconsuming strain, and a strain deficient for the ammonia transporter. Wild-type and metabolically engineered L. plantarum strains were administered in ornithine transcarbamoylase-deficient Sparse-fur mice, a model of constitutive hyperammonemia, in a carbon tetrachloride rat model of chronic liver insufficiency and in a thioacetamide-induced acute liver failure mice model. Constitutive hyperammonemia in Sparse-fur mice and hyperammonemia in a rat model of chronic hepatic insufficiency were efficiently decreased by Lactobacillus administration. In a murine thioacetamide-induced model of acute liver failure, administration of probiotics significantly increased survival and decreased blood and fecal ammonia. The ammonia hyperconsuming strain exhibited a beneficial effect at a lower dose than its wild-type counterpart. Improved survival in the acute liver failure mice model was associated with lower blood ammonia levels but also with a decrease of astrocyte swelling in the brain cortex. Modulation of ammonia was abolished after administration of the strain deficient in the ammonium transporter. Intestinal pH was clearly lowered for all strains and no changes in gut flora were observed. Hyperammonemia in constitutive model or after acute or chronic induced liver failure can be controlled by the administration of L. plantarum with a significant effect on survival. The mechanism involved in this ammonia decrease implicates direct ammonia consumption in the gut.

Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice.

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Kelly, Daniel M; Nettleship, Joanne E; Akhtar, Samia; Muraleedharan, Vakkat; Sellers, Donna J; Brooke, Jonathan C; McLaren, David S; Channer, Kevin S; Jones, T Hugh

2014-07-30

Non-alcoholic fatty liver disease and its precursor hepatic steatosis is common in obesity and type-2 diabetes and is associated with cardiovascular disease (CVD). Men with type-2 diabetes and/or CVD have a high prevalence of testosterone deficiency. Testosterone replacement improves key cardiovascular risk factors. The effects of testosterone on hepatic steatosis are not fully understood. Testicular feminised (Tfm) mice, which have a non-functional androgen receptor (AR) and very low serum testosterone levels, were used to investigate testosterone effects on high-cholesterol diet-induced hepatic steatosis. Hepatic lipid deposition was increased in Tfm mice and orchidectomised wild-type littermates versus intact wild-type littermate controls with normal androgen physiology. Lipid deposition was reduced in Tfm mice receiving testosterone treatment compared to placebo. Oestrogen receptor blockade significantly, but only partially, reduced the beneficial effects of testosterone treatment on hepatic lipid accumulation. Expression of key regulatory enzymes of fatty acid synthesis, acetyl-CoA carboxylase alpha (ACACA) and fatty acid synthase (FASN) were elevated in placebo-treated Tfm mice versus placebo-treated littermates and Tfm mice receiving testosterone treatment. Tfm mice on normal diet had increased lipid accumulation compared to littermates but significantly less than cholesterol-fed Tfm mice and demonstrated increased gene expression of hormone sensitive lipase, stearyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma but FASN and ACACA were not altered. An action of testosterone on hepatic lipid deposition which is independent of the classic AR is implicated. Testosterone may act in part via an effect on the key regulatory lipogenic enzymes to protect against hepatic steatosis. Copyright © 2014 Elsevier Inc. All rights reserved.

AMPKγ3 is dispensable for skeletal muscle hypertrophy induced by functional overload.

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Riedl, Isabelle; Osler, Megan E; Björnholm, Marie; Egan, Brendan; Nader, Gustavo A; Chibalin, Alexander V; Zierath, Juleen R

2016-03-15

Mechanisms regulating skeletal muscle growth involve a balance between the activity of serine/threonine protein kinases, including the mammalian target of rapamycin (mTOR) and 5'-AMP-activated protein kinase (AMPK). The contribution of different AMPK subunits to the regulation of cell growth size remains inadequately characterized. Using AMPKγ3 mutant-overexpressing transgenic Tg-Prkag3(225Q) and AMPKγ3-knockout (Prkag3(-/-)) mice, we investigated the requirement for the AMPKγ3 isoform in functional overload-induced muscle hypertrophy. Although the genetic disruption of the γ3 isoform did not impair muscle growth, control sham-operated AMPKγ3-transgenic mice displayed heavier plantaris muscles in response to overload hypertrophy and underwent smaller mass gain and lower Igf1 expression compared with wild-type littermates. The mTOR signaling pathway was upregulated with functional overload but unchanged between genetically modified animals and wild-type littermates. Differences in AMPK-related signaling pathways between transgenic, knockout, and wild-type mice did not impact muscle hypertrophy. Glycogen content was increased following overload in wild-type mice. In conclusion, our functional, transcriptional, and signaling data provide evidence against the involvement of the AMPKγ3 isoform in the regulation of skeletal muscle hypertrophy. Thus, the AMPKγ3 isoform is dispensable for functional overload-induced muscle growth. Mechanical loading can override signaling pathways that act as negative effectors of mTOR signaling and consequently promote skeletal muscle hypertrophy. Copyright © 2016 the American Physiological Society.

Intact interval timing in circadian CLOCK mutants.

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Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

A selective role for α3 subunit glycine receptors in inflammatory pain

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Victoria L Harvey

2009-11-01

Full Text Available GlyR α3 has previously been found to play a critical role in pain hypersensitivity following spinal PGE2 injection, complete Freund’s adjuvant (CFA and zymosan induced peripheral inflammation. In this study, although all models displayed typical phenotypic behaviours, no significant differences were observed when comparing the pain behaviours of Glra3-/- and wild-type littermates following the injection of capsaicin, carrageenan, kaolin/ carrageenan or monosodium iodoacetate, models of rheumatoid and osteoarthritis, respectively. However, clear differences were observed following CFA injection (p < 0.01. No significant differences were observed in the pain behaviours of Glra3-/- and wild-type littermates following experimentally induced neuropathic pain (partial sciatic nerve ligation. Similarly, Glra3-/- and wild-type littermates displayed indistinguishable visceromotor responses to colorectal distension (a model of visceral pain and in vivo spinal cord dorsal horn electrophysiology revealed no differences in responses to multimodal suprathreshold stimuli, intensities which equate to higher pain scores such as those reported in the clinic. These data suggest that apart from its clear role in CFA- and zymosan-induced pain sensitisation, hypersensitivity associated with other models of inflammation, neuropathy and visceral disturbances involves mechanisms other than the EP2 receptor - GlyR α3 pathway.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

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Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Growth deficits in cystic fibrosis mice begin in utero prior to IGF-1 reduction.

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Rebecca Darrah

Full Text Available Growth deficits are common in cystic fibrosis (CF, but their cause is complex, with contributions from exocrine pancreatic insufficiency, pulmonary complications, gastrointestinal obstructions, and endocrine abnormalities. The CF mouse model displays similar growth impairment despite exocrine pancreatic function and in the absence of chronic pulmonary infection. The high incidence of intestinal obstruction in the CF mouse has been suggested to significantly contribute to the observed growth deficits. Previous studies by our group have shown that restoration of the cystic fibrosis transmembrane conductance regulator (CFTR in the intestinal epithelium prevents intestinal obstruction but does not improve growth. In this study, we further investigate growth deficits in CF and gut-corrected CF mice by assessing insulin-like growth factor 1 (IGF-1. IGF-1 levels were significantly decreased in CF and gut-corrected CF adult mice compared to wildtype littermates and were highly correlated with weight. Interestingly, perinatal IGF-1 levels were not significantly different between CF and wildtype littermates, even though growth deficits in CF mice could be detected late in gestation. Since CFTR has been suggested to play a role in water and nutrient exchange in the placenta through its interaction with aquaporins, we analyzed placental aquaporin expression in late-gestation CF and control littermates. While significant differences were observed in Aquaporin 9 expression in CF placentas in late gestation, there was no evidence of placental fluid exchange differences between CF and control littermates. The results from this study indicate that decreased IGF-1 levels are highly correlated with growth in CF mice, independent of CF intestinal obstruction. However, the perinatal growth deficits that are observed in CF mice are not due to decreased IGF-1 levels or differences in placenta-mediated fluid exchange. Further investigation is necessary to understand

Aged dominant negative p38α MAPK mice are resistant to age-dependent decline in adult-neurogenesis and context discrimination fear conditioning.

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Cortez, IbDanelo; Bulavin, Dmitry V; Wu, Ping; McGrath, Erica L; Cunningham, Kathryn A; Wakamiya, Maki; Papaconstantinou, John; Dineley, Kelly T

2017-03-30

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38α AF/+ ) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38α AF/+ ) and kinase activity. As a result, aged DN-p38α AF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates. Aging is the major risk factor for non-inherited forms of Alzheimer's disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual's relative risk. In the present study, we evaluated aged DN-p38α AF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38α AF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38α AF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38α AF/+ , we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38α AF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of Lowering Asymmetric Dimethylarginine (ADMA on Vascular Pathology in Atherosclerotic ApoE-Deficient Mice with Reduced Renal Mass

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Johannes Jacobi

2014-03-01

Full Text Available The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS inhibitor asymmetric dimethylarginine (ADMA and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1, on atherosclerosis in subtotally nephrectomized (SNX ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n = 39 and C57Bl/6J wild-type littermates (WT, n = 27 with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction ApoE-competent DDAH1 transgenic and wild-type littermates (n = 11 served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD in this mouse model.

Structural Characterization of Lignin in Wild-Type versus COMT Down-Regulated Switchgrass

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Samuel, Reichel [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Pu, Yunqiao, E-mail: yunqiao.pu@ipst.gatech.edu [BioEnergy Science Center, Oak Ridge, TN (United States); Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA (United States); Jiang, Nan [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Fu, Chunxiang [Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Wang, Zeng-Yu [BioEnergy Science Center, Oak Ridge, TN (United States); Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Ragauskas, Arthur, E-mail: yunqiao.pu@ipst.gatech.edu [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States)

2014-01-20

This study examined the chemical structural characteristics of cellulolytic enzyme lignin isolated from switchgrass focusing on comparisons between wild-type control and caffeic acid 3-O-methyltransferase (COMT) down-regulated transgenic line. Nuclear magnetic resonance techniques including {sup 13}C, {sup 31}P, and two-dimensional {sup 13}C-{sup 1}H heteronuclear single quantum coherence as well as gel permeation chromatography were employed. Compared to the wild-type, the COMT down-regulated transgenic switchgrass lignin demonstrated a decrease in syringyl (S):guaiacyl (G) ratio and p-coumarate:ferulate ratio, an increase in relative abundance of phenylcoumaran unit, and a comparable content of total free phenolic OH groups along with formation of benzodioxane unit. In addition, COMT down-regulation had no significant effects on the lignin molecular weights during its biosynthesis process.

Quantification of amyloid deposits and oxygen extraction fraction in the brain with multispectral optoacoustic imaging in arcAβ mouse model of Alzheimer's disease

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Ni, Ruiqing; Vaas, Markus; Rudin, Markus; Klohs, Jan

2018-02-01

Beta-amyloid (Aβ) deposition and vascular dysfunction are important contributors to the pathogenesis in Alzheimer's disease (AD). However, the spatio-temporal relationship between an altered oxygen metabolism and Aβ deposition in the brain remains elusive. Here we provide novel in-vivo estimates of brain Aβ load with Aβ-binding probe CRANAD-2 and measures of brain oxygen saturation by using multi-spectral optoacoustic imaging (MSOT) and perfusion imaging with magnetic resonance imaging (MRI) in arcAβ mouse models of AD. We demonstrated a decreased cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) in the cortical region of the arcAβ mice compared to wildtype littermates at 24 months. In addition, we showed proof-of-concept for the detection of cerebral Aβ deposits in brain from arcAβ mice compared to wild-type littermates.

Interleukin 21 controls tumour growth and tumour immunosurveillance in colitis-associated tumorigenesis in mice.

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Jauch, Dominik; Martin, Maria; Schiechl, Gabriela; Kesselring, Rebecca; Schlitt, Hans Jürgen; Geissler, Edward K; Fichtner-Feigl, Stefan

2011-12-01

Colitis-associated tumorigenesis is a balance between proliferation of tumour cells and tumour immunosurveillance. The role of T-helper-cell-derived cytokines in tumour growth is not fully understood. In this study the authors investigated the influence of interleukin (IL) 21 on intestinal tumorigenesis. Chronic colitis was induced in IL-21(-/-) and littermate control wild-type mice with three cycles of 1.5% dextran sulphate sodium (DSS) over 7 days followed by 7 days of drinking water. Mice received an azoxymethane injection on day 0 of DSS-colitis to induce tumorigenesis. Immunohistochemistry was performed on inflamed and tumour-bearing areas of colons. Cytokine expression of isolated colonic CD4 T cells was determined by ELISA. Cytotoxic capacity of isolated colonic CD8 T cells targeting tumour cells was evaluated by flow cytometry and quantitative cytotoxicity assay. Apoptosis of tumour cells was determined by TUNEL assay of colonic sections. Increasing expression of IL-21 was observed in chronic colitis, which showed functional importance, since IL-21 deficiency prevented chronic DSS-colitis development. Further, in the absence of IL-21, significantly fewer tumour nodules were detected, despite a similar extent of intestinal inflammation. In wild-type mice, 8.6±1.9 tumour nodules were found compared with 1.0±1.2 in IL-21-deficient mice. In tumour-bearing IL-21-deficient mice, intestinal inflammation was restored and partly dependent on interferon (IFN)-γ, whereas the inflammation in wild-type mice showed high IL-17A concentrations. In these rare tumours in IL-21-deficient mice, tumour cell proliferation (Ki-67) was decreased, while cell apoptosis was increased, compared with wild-type mice. Increased IFNγ expression in tumour-bearing IL-21-deficient mice led to increased tumour immunosurveillance mediated by cytotoxic CD8CD103 T cells targeting E-cadherin(+) colonic tumour cells and therefore limited tumour growth. These results indicate that IL-21

Nfib hemizygous mice are protected from hyperoxic lung injury and death.

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Kumar, Vasantha H S; Chaker El Khoury, Joseph; Gronostajski, Richard; Wang, Huamei; Nielsen, Lori; Ryan, Rita M

2017-08-01

Nuclear Factor I ( Nfi) genes encode transcription factors essential for the development of organ systems including the lung. Nfib null mice die at birth with immature lungs. Nfib hemizygous mice have reduced lung maturation with decreased survival. We therefore hypothesized that these mice would be more sensitive to lung injury and would have lower survival to hyperoxia. Adult Nfib hemizygous mice and their wild-type (Wt) littermates were exposed to 100% O 2 for 89, 80, 72 and 66 h for survival studies with lung outcome measurements at 66 h. Nfib hemizygous and Wt controls were also studied in RA at 66 h. Cell counts and cytokines were measured in bronchoalveolar lavage (BAL); lung sections examined by histopathology; lung angiogenic and oxidative stress gene expression assessed by real-time PCR Unexpectedly, Nfib hemizygous mice (0/14-0%) had significantly lower mortality compared to Wt mice (10/22-45%) at 80 h of hyperoxia ( P  mice exposed to hyperoxia. New vessel formation, edema, congestion, and alveolar hemorrhage were noted on histopathology at 72 and 80 h in wild-type mice. Nfib hemizygous lungs had significant downregulation of genes involved in redox signaling and inflammatory pathways. Adult Nfib hemizygous mice are relatively resistant to hyperoxia compared to wild-type littermates. Mechanisms contributing to this resistance are not clear; however, transcription factors such as Nfib may regulate cell survival and play a role in modulating postnatal lung development. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B

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Walz, Helena A; Härndahl, Linda; Wierup, Nils

2006-01-01

Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether beta-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a beta-cell-specific, 2-fold overexpression of the c......AMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from...... did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet...

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

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Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

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Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun

2003-10-01

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

The In Vivo Granulopoietic Response to Dexamethasone Injection Is Abolished in Perforin-Deficient Mutant Mice and Corrected by Lymphocyte Transfer from Nonsensitized Wild-Type Donors

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Pedro Xavier-Elsas

2015-01-01

Full Text Available Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp- deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.

PGC-1alpha is required for training-induced prevention of age-associated decline in mitochondrial enzymes in mouse skeletal muscle

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Leick, Lotte; Lyngby, Stine Secher; Wojtaszewski, Jørgen

2010-01-01

The aim of the present study was to test the hypothesis that exercise training prevents an age-associated decline in skeletal muscle mitochondrial enzymes through a PGC-1alpha dependent mechanism. Whole body PGC-1alpha knock-out (KO) and littermate wildtype (WT) mice were submitted to long term...

Apolipoprotein C3 deficiency results in diet-induced obesity and aggravated insulin resistance in mice

NARCIS (Netherlands)

Duivenvoorden, Ilse; Teusink, Bas; Rensen, Patrick C.; Romijn, Johannes A.; Havekes, Louis M.; Voshol, Peter J.

2005-01-01

Our aim was to study whether the absence of apolipoprotein (apo) C3, a strong inhibitor of lipoprotein lipase (LPL), accelerates the development of obesity and consequently insulin resistance. Apoc3(-/-) mice and wild-type littermates were fed a high-fat (46 energy %) diet for 20 weeks. After 20

Skin wound healing in MMP2-deficient and MMP2 / plasminogen double-deficient mice

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Frøssing, Signe; Rønø, Birgitte; Hald, Andreas

2010-01-01

-sensitive MMPs during wound healing. To address whether MMP2 is accountable for the galardin-induced healing deficiency in wildtype and Plg-deficient mice, incisional skin wounds were generated in MMP2 single-deficient mice and in MMP2/Plg double-deficient mice and followed until healed. Alternatively, tissue...... was isolated 7 days post wounding for histological and biochemical analyses. No difference was found in the time from wounding to overt gross restoration of the epidermal surface between MMP2-deficient and wildtype control littermate mice. MMP2/Plg double-deficient mice were viable and fertile, and displayed...... an unchallenged general phenotype resembling that of Plg-deficient mice, including development of rectal prolapses. MMP2/Plg double-deficient mice displayed a slight increase in the wound length throughout the healing period compared with Plg-deficient mice. However, the overall time to complete healing...

Perception of sweet taste is important for voluntary alcohol consumption in mice.

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Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F

2008-02-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

Anthelmintic effect of Psidium guajava and Tagetes erecta on wild-type and Levamisole-resistant Caenorhabditis elegans strains.

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Piña-Vázquez, Denia M; Mayoral-Peña, Zyanya; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A; Arellano-Carbajal, Fausto

2017-04-18

Psidium guajava and Tagetes erecta have been used traditionally to treat gastrointestinal parasites, but their active metabolites and mechanisms of action remain largely unknown. To evaluate the anthelmintic potential of Psidium guajava and Tagetes erecta extracts on Levamisole-sensitive and Levamisole-resistant strains of the model nematode Caenorhabditis elegans. Aqueous extracts of Psidium guajava (PGE) and Tagetes erecta (TEE) were assayed on locomotion and egg-laying behaviors of the wild-type (N2) and Levamisole-resistant (CB193) strains of Caenorhabditis elegans. Both extracts paralyzed wild-type and Levamisole-resistant nematodes in a dose-dependent manner. In wild-type worms, TEE 25mg/mL induced a 75% paralysis after 8h of treatment and PGE 25mg/mL induced a 100% paralysis after 4h of treatment. PGE exerted a similar paralyzing effect on N2 wild-type and CB193 Levamisole-resistant worms, while TEE only partially paralyzed CB193 worms. TEE 25mg/mL decreased N2 egg-laying by 65% with respect to the untreated control, while PGE did it by 40%. Psidium guajava leaves and Tagetes erecta flower-heads possess hydrosoluble compounds that block the motility of Caenorhabditis elegans by a mechanism different to that of the anthelmintic drug Levamisole. Effects are also observable on oviposition, which was diminished in the wild-type worms. The strong anthelmintic effects in crude extracts of these plants warrants future work to identify their active compounds and to elucidate their molecular mechanisms of action. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling

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Greenhalgh, Christopher J; Metcalf, Donald; Thaus, Anne L

2002-01-01

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may at...

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

DEFF Research Database (Denmark)

Jensen, T G; Andresen, B S; Bross, P

1992-01-01

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

Neurochemical and behavioral characterization of neuronal glutamate transporter EAAT3 heterozygous mice

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Luis F. González

2017-10-01

Full Text Available Abstract Background Obsessive–compulsive disorder (OCD is a severe neuropsychiatric condition affecting 1–3% of the worldwide population. OCD has a strong genetic component, and the SLC1A1 gene that encodes neuronal glutamate transporter EAAT3 is a strong candidate for this disorder. To evaluate the impact of reduced EAAT3 expression in vivo, we studied male EAAT3 heterozygous and wild-type littermate mice using a battery of behavioral paradigms relevant to anxiety (open field test, elevated plus maze and compulsivity (marble burying, as well as locomotor activity induced by amphetamine. Using high-performance liquid chromatography, we also determined tissue neurotransmitter levels in cortex, striatum and thalamus—brain areas that are relevant to OCD. Results Compared to wild-type littermates, EAAT3 heterozygous male mice have unaltered baseline anxiety-like, compulsive-like behavior and locomotor activity. Administration of acute amphetamine (5 mg/kg intraperitoneally increased locomotion with no differences across genotypes. Tissue levels of glutamate, GABA, dopamine and serotonin did not vary between EAAT3 heterozygous and wild-type mice. Conclusions Our results indicate that reduced EAAT3 expression does not impact neurotransmitter content in the corticostriatal circuit nor alter anxiety or compulsive-like behaviors.

Activation Of Wild-Type Hras Suppresses The Earliest Stages Of Pancreatic Cancer

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Jamie Weyandt

2015-08-01

Conclusions: Loss of wild-type Hras promotes the earliest stages of pancreatic tumorigenesis, and moreover results in more rapid progression of the disease. As such, mechanisms leading to activation of wild-type Ras proteins, including but not limited to redox-dependent reactions, may influence the development of pancreatic cancer.

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

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Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

2014-05-21

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

Wfs1-deficient mice display altered function of serotonergic system and increased behavioural response to antidepressants

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Tanel eVisnapuu

2013-07-01

Full Text Available It has been shown that mutations in the WFS1 gene make humans more susceptible to mood disorders. Besides that, mood disorders are associated with alterations in the activity of serotonergic and noradrenergic systems. Therefore, in this study, the effects of imipramine, an inhibitor of serotonin (5-HT and noradrenaline (NA reuptake, and paroxetine, a selective inhibitor of 5-HT reuptake, were studied in tests of behavioural despair. The tail suspension test (TST and forced swimming test (FST were performed in Wfs1-deficient mice. Simultaneously, gene expression and monoamine metabolism studies were conducted to evaluate changes in 5-HT- and NA-ergic systems of Wfs1-deficient mice. The basal immobility time of Wfs1-deficient mice in TST and FST did not differ from that of their wild-type littermates. However, a significant reduction of immobility time in response to lower doses of imipramine and paroxetine was observed in homozygous Wfs1-deficient mice, but not in their wild-type littermates. In gene expression studies, the levels of 5-HT transporter (SERT were significantly reduced in the pons of homozygous animals. Monoamine metabolism was assayed separately in the dorsal and ventral striatum of naive mice and mice exposed for 30 minutes tobrightly lit motility boxes. We found that this aversive challenge caused a significant increase in the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA, a metabolite of 5-HT, in the ventral and dorsal striatum of wild-type mice, but not in their homozygous littermates. Taken together, the blunted 5-HT metabolism and reduced levels of SERT are a likely reason for the elevated sensitivity of these mice to the action of imipramine and paroxetine. These changes in the pharmacological and neurochemical phenotype of Wfs1-deficient mice may help to explain the increased susceptibility of Wolfram syndrome patients to depressive states.

Protein tyrosine phosphatase 1B deficiency ameliorates murine experimental colitis via the expansion of myeloid-derived suppressor cells.

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Jing Zhang

Full Text Available Protein tyrosine phosphatase 1B (PTP1B is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS-induced murine experimental colitis via expanding CD11b(+Gr-1(+ myeloid-derived suppressor cells (MDSCs. Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM, peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3 and Janus kinase 2 (JAK2. In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.

Mice deleted for cell division cycle 73 gene develop parathyroid and uterine tumours: model for the hyperparathyroidism-jaw tumour syndrome.

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Walls, G V; Stevenson, M; Lines, K E; Newey, P J; Reed, A A C; Bowl, M R; Jeyabalan, J; Harding, B; Bradley, K J; Manek, S; Chen, J; Wang, P; Williams, B O; Teh, B T; Thakker, R V

2017-07-13

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by occurrence of parathyroid tumours, often atypical adenomas and carcinomas, ossifying jaw fibromas, renal tumours and uterine benign and malignant neoplasms. HPT-JT is caused by mutations of the cell division cycle 73 (CDC73) gene, located on chromosome 1q31.2 and encodes a 531 amino acid protein, parafibromin. To facilitate in vivo studies of Cdc73 in tumourigenesis we generated conventional (Cdc73 +/- ) and conditional parathyroid-specific (Cdc73 +/L /PTH-Cre and Cdc73 L/L /PTH-Cre) mouse models. Mice were aged to 18-21 months and studied for survival, tumour development and proliferation, and serum biochemistry, and compared to age-matched wild-type (Cdc73 +/+ and Cdc73 +/+ /PTH-Cre) littermates. Survival of Cdc73 +/- mice, when compared to Cdc73 +/+ mice was reduced (Cdc73 +/- =80%; Cdc73 +/+ =90% at 18 months of age, Pfourfold higher than that in parathyroid glands of wild-type littermates (P<0.0001). Cdc73 +/- , Cdc73 +/L /PTH-Cre and Cdc73 L/L /PTH-Cre mice had higher mean serum calcium concentrations than wild-type littermates, and Cdc73 +/- mice also had increased mean serum parathyroid hormone (PTH) concentrations. Parathyroid tumour development, and elevations in serum calcium and PTH, were similar in males and females. Cdc73 +/- mice did not develop bone or renal tumours but female Cdc73 +/- mice, at 18 months of age, had uterine neoplasms comprising squamous metaplasia, adenofibroma and adenomyoma. Uterine neoplasms, myometria and jaw bones of Cdc73 +/- mice had increased proliferation rates that were 2-fold higher than in Cdc73 +/+ mice (P<0.05). Thus, our studies, which have established mouse models for parathyroid tumours and uterine neoplasms that develop in the HPT-JT syndrome, provide in vivo models for future studies of these tumours.

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan.

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Rashid, Harunur; Chen, Haiyan; Hassan, Quamarul; Javed, Amjad

2017-10-01

Aggrecan (Acan), a large proteoglycan is abundantly expressed in cartilage tissue. Disruption of Acan gene causes dwarfism and perinatal lethality of homozygous mice. Because of sustained expression of Acan in the growth plate and articular cartilage, Agc Cre model has been developed for the regulated ablation of target gene in chondrocytes. In this model, the IRES-CreERT-Neo-pgk transgene is knocked-in the 3'UTR of the Acan gene. We consistently noticed variable weight and size among the Agc Cre littermates, prompting us to examine the cause of this phenotype. Wild-type, Cre-heterozygous (Agc +/Cre ), and Cre-homozygous (Agc Cre/Cre ) littermates were indistinguishable at birth. However, by 1-month, Agc Cre/Cre mice showed a significant reduction in body weight (18-27%) and body length (19-22%). Low body weight and dwarfism was sustained through adulthood and occurred in both genders. Compared with wild-type and Agc +/Cre littermates, long bones and vertebrae were shorter in Agc Cre/Cre mice. Histological analysis of Agc Cre/Cre mice revealed a significant reduction in the length of the growth plate and the thickness of articular cartilage. The amount of proteoglycan deposited in the cartilage of Agc Cre/Cre mice was nearly half of the WT littermates. Analysis of gene expression indicates impaired differentiation of chondrocyte in hyaline cartilage of Agc Cre/Cre mice. Notably, both Acan mRNA and protein was reduced by 50% in Agc Cre/Cre mice. A strong correlation was noted between the level of Acan mRNA and the body length. Importantly, Agc +/Cre mice showed no overt skeletal phenotype. Thus to avoid misinterpretation of data, only the Agc +/Cre mice should be used for conditional deletion of a target gene in the cartilage tissue. © 2017 Wiley Periodicals, Inc.

Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

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Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

2009-02-01

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

Near-Infrared Spectroscopy, a Rapid Method for Predicting the Age of Male and Female Wild-Type and Wolbachia Infected Aedes aegypti.

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Maggy T Sikulu-Lord

2016-10-01

Full Text Available Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (P< 0.001 in both instances. The age of wild-type female Ae. aegypti could be identified as < or ≥ 8 days old with an accuracy of 91% (N = 501, whereas female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284 and 78% (N = 229, respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253, 83% (N = 277 and 78% (N = 234, respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs.

Mammary tumorigenesis in APC{sup min/+} mice is enhanced by X-irradiation with a characteristic age dependence

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Tatsuhiko, Imaoka; Mayumi, Nishimura; Shizuko, Kakinuma; Yoshiya, Shimada [National Institute of Radiological Sciences, Experimental Radiobiology for Children' s Health Research Group, Research, Center for Radiation Protection (Japan); Mieko, Okamoto [Tokyo Metropolitan Institute of Medical Science (Japan)

2006-07-01

The ApcM{sup min/+} (Min) mouse is a genetically predisposed model of both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X-rays at 2, 5, 7 and 10 weeks and sacrificed at 18 weeks of age. Min mice irradiated at 7 to 10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type litter-mates did not. Interestingly, irradiation of Min mice at 2 to 5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling. (author)

Mammary tumorigenesis in APCmin/+ mice is enhanced by X-irradiation with a characteristic age dependence

International Nuclear Information System (INIS)

Tatsuhiko, Imaoka; Mayumi, Nishimura; Shizuko, Kakinuma; Yoshiya, Shimada; Mieko, Okamoto

2006-01-01

The ApcM min/+ (Min) mouse is a genetically predisposed model of both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X-rays at 2, 5, 7 and 10 weeks and sacrificed at 18 weeks of age. Min mice irradiated at 7 to 10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type litter-mates did not. Interestingly, irradiation of Min mice at 2 to 5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling. (author)

AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

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Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

2014-01-01

Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

Differentiation between probiotic and wild-type Bacillus cereus isolates by antibiotic susceptibility test and Fourier transform infrared spectroscopy (FT-IR).

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Mietke, Henriette; Beer, W; Schleif, Julia; Schabert, G; Reissbrodt, R

2010-05-30

Animal feed often contains probiotic Bacillus strains used as feed additives. Spores of the non-pathogenic B. cereus var. toyoi (product name Toyocerin) are used. Distinguishing between toxic wild-type Bacillus cereus strains and this probiotic strain is essential for evaluating the quality and risk of feed. Bacillus cereus CIP 5832 (product name Paciflor was used as probiotic strain until 2001. The properties of the two probiotic strains are quite similar. Differentiating between probiotic strains and wild-type B. cereus strains is not easy. ss-lactam antibiotics such as penicillin and cefamandole exhibit an inhibition zone in the agar diffusion test of probiotic B. cereus strains which are not seen for wild-type strains. Therefore, performing the agar diffusion test first may make sense before FT-IR testing. When randomly checking these strains by Fourier transform infrared spectroscopy (FT-IR), the probiotic B. cereus strains were separated from wild-type B. cereus/B. thuringiensis/B. mycoides/B. weihenstephanensis strains by means of hierarchical cluster analysis. The discriminatory information was contained in the spectral windows 3000-2800 cm(-1) ("fatty acid region"), 1200-900 cm(-1) ("carbohydrate region") and 900-700 cm(-1) ("fingerprint region"). It is concluded that FT-IR spectroscopy can be used for the rapid quality control and risk analysis of animal feed containing probiotic B. cereus strains. (c) 2010 Elsevier B.V. All rights reserved.

Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

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Yoshiaki Furukawa

2012-01-01

Full Text Available Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1 gene cause a familial form of amyotrophic lateral sclerosis (ALS. While it remains controversial how SOD1 mutations lead to onset and progression of the disease, many in vitro and in vivo studies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations in sod1 gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs.

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Lluís Sánchez

Full Text Available Four full-sibling intact male Miniature Poodles were evaluated at 4-19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog.

Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1α and HIF-2α

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Yoon, Donghoon; Okhotin, David V.; Kim, Bumjun; Okhotina, Yulia; Okhotin, Daniel J.; Miasnikova, Galina Y.; Sergueeva, Adelina I.; Polyakova, Lydia A.; Maslow, Alexei; Lee, Yonggu; Semenza, Gregg L.; Prchal, Josef T.

2010-01-01

Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHLR200W) resulting in elevated hypoxia inducible factor (HIF)-1α and HIF-2α levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21Cip1, thereby inducing its expression. In contrast, HIF-2α promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a+/− mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1α, hepatic HIF-2α mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21Cip1 mRNA levels were 9.5-fold lower in Hif1a+/− mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2α and decreased p21Cip1 levels leading to increased hepatocyte proliferation. PMID:20140661

A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

Science.gov (United States)

Witherow, D. Scott

2016-01-01

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

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Victoria Matyushenko

Full Text Available Live attenuated influenza vaccines (LAIVs are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

Altered ingestive behavior, weight changes, and intact olfactory sense in an APP overexpression model.

Science.gov (United States)

Vloeberghs, Ellen; Van Dam, Debby; Franck, Frieda; Serroyen, Jan; Geert, Molenberghs; Staufenbiel, Matthias; De Deyn, Peter Paul

2008-06-01

Transgenic APP23 mice were generated to model Alzheimer's disease. The APP23 model develops pathological features, learning deficits, and memory deficits analogous to dementing patients. In this report, transgenic mice exhibited several behavioral disturbances indicating the presence of neuropsychiatric symptoms of dementia. Aiming to verify whether the model also develops other behavioral problems, the authors investigated ingestive behavior in APP23 males of 3, 6 and 12 months. In addition, body weights of a naive male group were longitudinally monitored starting at weaning. Olfactory acuity was evaluated in mice of different age groups. Although olfactory functioning of APP23 mice appeared intact, they drank more and took more food pellets compared with wild-type littermates during a 1-week registration period. From the age of 4.5 weeks onward, APP23 males weighed significantly less than their control littermates, whereas this difference became more prominent with increasing age. Our results suggest the presence of a hypermetabolic state in this model. This is the first report, evidencing the presence of changes in eating and drinking behavior in a single transgenic Alzheimer mouse model. (Copyright) 2008 APA, all rights reserved.

Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities.

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Garrett, Matthew; Sperry, Jantzen; Braas, Daniel; Yan, Weihong; Le, Thuc M; Mottahedeh, Jack; Ludwig, Kirsten; Eskin, Ascia; Qin, Yue; Levy, Rachelle; Breunig, Joshua J; Pajonk, Frank; Graeber, Thomas G; Radu, Caius G; Christofk, Heather; Prins, Robert M; Lai, Albert; Liau, Linda M; Coppola, Giovanni; Kornblum, Harley I

2018-01-01

There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13 C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable

Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

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Morvan, Maelig G; Champsaur, Marine; Reizis, Boris; Lanier, Lewis L

2017-05-01

To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand-expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk.

Mice Expressing a "Hyper-Sensitive" Form of the Cannabinoid Receptor 1 (CB1 Are Neither Obese Nor Diabetic.

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David J Marcus

Full Text Available Multiple lines of evidence implicate the endocannabinoid signaling system in the modulation of metabolic disease. Genetic or pharmacological inactivation of CB1 in rodents leads to reduced body weight, resistance to diet-induced obesity, decreased intake of highly palatable food, and increased energy expenditure. Cannabinoid agonists stimulate feeding in rodents and increased levels of endocannabinoids can disrupt lipid metabolism. Therefore, the hypothesis that sustained endocannabinoid signaling can lead to obesity and diabetes was examined in this study using S426A/S430A mutant mice expressing a desensitization-resistant CB1 receptor. These mice display exaggerated and prolonged responses to acute administration of phytocannabinoids, synthetic cannabinoids, and endocannabinoids. As a consequence these mice represent a novel model for determining the effect of enhanced endocannabinoid signaling on metabolic disease. S426A/S430A mutants consumed equivalent amounts of both high fat (45% and low fat (10% chow control diet compared to wild-type littermate controls. S426A/S430A mutants and wild-type mice fed either high or low fat control diet displayed similar fasting blood glucose levels and normal glucose clearance following a 2 g/kg glucose challenge. Furthermore, S426A/S430A mutants and wild-type mice consumed similar amounts of chow following an overnight fast. While both THC and JZL195 significantly increased food intake two hours after injection, this increase was similar between the S426A/S430A mutant and wildtype control mice Our results indicate that S426A/S430A mutant mice expressing the desensitization-resistant form of CB1 do not exhibit differences in body weight, food intake, glucose homeostasis, or re-feeding following a fast.

Rearing in seawater mesocosms improves the spawning performance of growth hormone transgenic and wild-type coho salmon.

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Rosalind A Leggatt

Full Text Available Growth hormone (GH transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination has not been fully ascertained. Hence, salmon were reared in large (350,000 L, semi-natural, seawater tanks (termed mesocosms designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic

Unusual metabolic characteristics in skeletal muscles of transgenic rabbits for human lipoprotein lipase

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Viglietta Céline

2004-12-01

Full Text Available Abstract Background The lipoprotein lipase (LPL hydrolyses circulating triacylglycerol-rich lipoproteins. Thereby, LPL acts as a metabolic gate-keeper for fatty acids partitioning between adipose tissue for storage and skeletal muscle primarily for energy use. Transgenic mice that markedly over-express LPL exclusively in muscle, show increases not only in LPL activity, but also in oxidative enzyme activities and in number of mitochondria, together with an impaired glucose tolerance. However, the role of LPL in intracellular nutrient pathways remains uncertain. To examine differences in muscle nutrient uptake and fatty acid oxidative pattern, transgenic rabbits harboring a DNA fragment of the human LPL gene (hLPL and their wild-type littermates were compared for two muscles of different metabolic type, and for perirenal fat. Results Analyses of skeletal muscles and adipose tissue showed the expression of the hLPL DNA fragment in tissues of the hLPL group only. Unexpectedly, the activity level of LPL in both tissues was similar in the two groups. Nevertheless, mitochondrial fatty acid oxidation rate, measured ex vivo using [1-14C]oleate as substrate, was lower in hLPL rabbits than in wild-type rabbits for the two muscles under study. Both insulin-sensitive glucose transporter GLUT4 and muscle fatty acid binding protein (H-FABP contents were higher in hLPL rabbits than in wild-type littermates for the pure oxidative semimembranosus proprius muscle, but differences between groups did not reach significance when considering the fast-twitch glycolytic longissimus muscle. Variations in both glucose uptake potential, intra-cytoplasmic binding of fatty acids, and lipid oxidation rate observed in hLPL rabbits compared with their wild-type littermates, were not followed by any modifications in tissue lipid content, body fat, and plasma levels in energy-yielding metabolites. Conclusions Expression of intracellular binding proteins for both fatty acids and

Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses

International Nuclear Information System (INIS)

Burke, Crystal W.; Gardner, Christina L.; Steffan, Joshua J.; Ryman, Kate D.; Klimstra, William B.

2009-01-01

We examined the characteristics of interferon alpha/beta (IFN-α/β) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-α/β. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-α/β. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-β promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-α/β in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection.

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

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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.

Fusarium spp. Associated with Field-Grown Grain of Near-Isogenic Low Lignin and Wild-Type Sorghum

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Fusarium spp. associated with field-grown grain of near-isogenic low lignin and wild-type sorghum. Deanna Funnell-Harris and Jeff Pedersen, USDA-ARS, Lincoln, NE Previous studies indicated that low lignin brown midrib (bmr) sorghum may be more resistant to Fusarium spp. than wild-type and that phen...

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

NARCIS (Netherlands)

Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.

2009-01-01

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven

Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice.

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Wang, Jian; Menchenton, Trevor; Yin, Shankai; Yu, Zhiping; Bance, Manohar; Morris, David P; Moore, Craig S; Korneluk, Robert G; Robertson, George S

2010-07-01

Apoptosis of cochlear cells plays a significant role in age-related hearing loss or presbycusis. In this study, we evaluated whether over-expression of the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) slows the development of presbycusis. We compared the age-related hearing loss between transgenic (TG) mice that over-express human XIAP tagged with 6-Myc (Myc-XIAP) on a pure C57BL/6J genetic background with wild-type (WT) littermates by measuring auditory brainstem responses. The result showed that TG mice developed hearing loss considerably more slowly than WT littermates, primarily within the high-frequency range. The average total hair cell loss was significantly less in TG mice than WT littermates. Although levels of Myc-XIAP in the ear remained constant at 2 and 14 months, there was a marked increase in the amount of endogenous XIAP from 2 to 14 months in the cochlea, but not in the brain, in both genotypes. These results suggest that XIAP over-expression reduces age-related hearing loss and hair cell death in the cochlea. Copyright 2008 Elsevier Inc. All rights reserved.

DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

International Nuclear Information System (INIS)

Cuthill, S.; Poellinger, L.

1988-01-01

The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

Restoration of Haemoglobin Level Using Hydrodynamic Gene Therapy with Erythropoietin Does Not Alleviate the Disease Progression in an Anaemic Mouse Model for TGFβ1-Induced Chronic Kidney Disease

DEFF Research Database (Denmark)

Pedersen, Lea Hougaard; Wogensen, Lise; Marcussen, N.

2015-01-01

. The experiment is conducted by hydrodynamic gene transfer of a plasmid encoding murine Epo in a transgenic mouse model that overexpresses TGF-β1 locally in the kidneys. This model develops anaemia due to chronic kidney disease characterised by thickening of the glomerular basement membrane, deposition...... of mesangial matrix and mild interstitial fibrosis. A group of age matched wildtype littermates are treated accordingly. After a single hydrodynamic administration of plasmid DNA containing murine EPO gene, sustained high haemoglobin levels are observed in both transgenic and wildtype mice from 7.5 ± 0.6 mmol...... treatment in this model of chronic kidney disease normalises haemoglobin levels but has no effect on kidney fibrosis or function....

Evidence that insulin-like growth factor I and growth hormone are required for prostate gland development.

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Ruan, W; Powell-Braxton, L; Kopchick, J J; Kleinberg, D L

1999-05-01

Insulin-like growth factor I (IGF-I) has been implicated as a factor that may predispose one to prostate cancer. However, no specific relationship between IGF-I and prostate development or cancer in vivo has been established. To determine whether IGF-I was important in prostate development, we examined prostate architecture in IGF-I(-/-) null mice and wild-type littermates. Glands from 44-day-old IGF-I-deficient animals were not only smaller than those from wild-type mice, but also had fewer terminal duct tips and branch points and deficits in tertiary and quaternary branching (P deficit by increasing those parameters of prostate development (P growth as an extension of a normal process.

Blockade of dopamine D1-family receptors attenuates the mania-like hyperactive, risk-preferring, and high motivation behavioral profile of mice with low dopamine transporter levels.

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Milienne-Petiot, Morgane; Groenink, Lucianne; Minassian, Arpi; Young, Jared W

2017-10-01

Patients with bipolar disorder mania exhibit poor cognition, impulsivity, risk-taking, and goal-directed activity that negatively impact their quality of life. To date, existing treatments for bipolar disorder do not adequately remediate cognitive dysfunction. Reducing dopamine transporter expression recreates many bipolar disorder mania-relevant behaviors (i.e. hyperactivity and risk-taking). The current study investigated whether dopamine D 1 -family receptor blockade would attenuate the risk-taking, hypermotivation, and hyperactivity of dopamine transporter knockdown mice. Dopamine transporter knockdown and wild-type littermate mice were tested in mouse versions of the Iowa Gambling Task (risk-taking), Progressive Ratio Breakpoint Test (effortful motivation), and Behavioral Pattern Monitor (activity). Prior to testing, the mice were treated with the dopamine D 1 -family receptor antagonist SCH 23390 hydrochloride (0.03, 0.1, or 0.3 mg/kg), or vehicle. Dopamine transporter knockdown mice exhibited hyperactivity and hyperexploration, hypermotivation, and risk-taking preference compared with wild-type littermates. SCH 23390 hydrochloride treatment decreased premature responding in dopamine transporter knockdown mice and attenuated their hypermotivation. SCH 23390 hydrochloride flattened the safe/risk preference, while reducing activity and exploratory levels of both genotypes similarly. Dopamine transporter knockdown mice exhibited mania-relevant behavior compared to wild-type mice. Systemic dopamine D 1 -family receptor antagonism attenuated these behaviors in dopamine transporter knockdown, but not all effects were specific to only the knockdown mice. The normalization of behavior via blockade of dopamine D 1 -family receptors supports the hypothesis that D 1 and/or D 5 receptors could contribute to the mania-relevant behaviors of dopamine transporter knockdown mice.

Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

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Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

2009-06-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

Homozygous Wildtype of XPD K751Q Polymorphism Is Associated with Increased Risk of Nasopharyngeal Carcinoma in Malaysian Population.

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Munn-Sann Lye

Full Text Available The xeroderma pigmentosum group D (XPD gene encodes a DNA helicase, an important component in transcription factor IIH (TFIIH complex. XPD helicase plays a pivotal role in unwinding DNA at the damaged region during nucleotide excision repair (NER mechanism. Dysfunctional XPD helicase protein from polymorphic diversity may contribute to increased risk of developing cancers. This study aims to determine the association between XPD K751Q polymorphism (rs13181 and risk of nasopharyngeal carcinoma (NPC in the Malaysian population. In this hospital-based matched case-control study, 356 controls were matched by age, gender and ethnicity to 356 cases. RFLP-PCR was used to genotype the XPD K751Q polymorphism. A significant association was observed between XPD K751Q polymorphism and the risk of NPC using conditional logistic regression. Subjects with homozygous Lys/Lys (wildtype genotype have 1.58 times higher odds of developing NPC compared to subjects with recessive combination of heterozygous Lys/Gln and homozygous Gln/Gln genotypes (OR = 1.58, 95% CI = 1.05-2.38 p = 0.028 adjusted for cigarette smoking, alcohol and salted fish consumption. Our data suggests that Lys/Lys (wildtype of XPD K751Q contributes to increased risk of NPC in the Malaysian population.

Osteoblast-targeted overexpression of TAZ increases bone mass in vivo.

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Jae-Yeon Yang

Full Text Available Osteoblasts are derived from mesenchymal progenitors. Differentiation to osteoblasts and adipocytes is reciprocally regulated. Transcriptional coactivator with a PDZ-binding motif (TAZ is a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts while blocking differentiation into adipocytes. To investigate the role of TAZ on bone metabolism in vivo, we generated transgenic mice that overexpress TAZ under the control of the procollagen type 1 promoter (Col1-TAZ. Whole body bone mineral density (BMD of 6- to 19-week-old Col-TAZ mice was 4% to 7% higher than that of their wild-type (WT littermates, whereas no difference was noticed in Col.1-TAZ female mice. Microcomputed tomography analyses of proximal tibiae at 16 weeks of age demonstrated a significant increase in trabecular bone volume (26.7% and trabecular number (26.6% with a reciprocal decrease in trabecular spacing (14.2% in Col1-TAZ mice compared with their WT littermates. In addition, dynamic histomorphometric analysis of the lumbar spine revealed increased mineral apposition rate (42.8% and the serum P1NP level was also significantly increased (53% in Col.1-TAZ mice. When primary calvaria cells were cultured in osteogenic medium, alkaline phosphatase (ALP activity was significantly increased and adipogenesis was significantly suppressed in Col1-TAZ mice compared with their WT littermates. Quantitative real-time polymerase chain reaction analyses showed that expression of collagen type 1, bone sialoprotein, osteocalcin, ALP, osterix, and Runx2 was significantly increased in calvaria cells from Col1-TAZ mice compared to their WT littermates. In vitro, TAZ enhanced Runx2-mediated transcriptional activity while suppressing the peroxisome proliferator-activated receptor gamma signaling pathway. TAZ also enhanced transcriptional activity from 3TP-Lux, which reflects transforming growth factor-beta (TGF-β-mediated signaling. In addition, TAZ enhanced TGF

Identifying the Integrated Neural Networks Involved in Capsaicin-Induced Pain Using fMRI in Awake TRPV1 Knockout and Wild-Type Rats

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Jason Richard Yee

2015-02-01

Full Text Available In the present study, we used functional MRI in awake rats to investigate the pain response that accompanies intradermal injection of capsaicin into the hindpaw. To this end, we used BOLD imaging together with a 3D segmented, annotated rat atlas and computational analysis to identify the integrated neural circuits involved in capsaicin-induced pain. The specificity of the pain response to capsaicin was tested in a transgenic model that contains a biallelic deletion of the gene encoding for the transient receptor potential cation channel subfamily V member 1 (TRPV1. Capsaicin is an exogenous ligand for the TRPV1 receptor, and in wild-type rats, activated the putative pain neural circuit. In addition, capsaicin-treated wild-type rats exhibited activation in brain regions comprising the Papez circuit and habenular system, systems that play important roles in the integration of emotional information, and learning and memory of aversive information, respectively. As expected, capsaicin administration to TRPV1-KO rats failed to elicit the robust BOLD activation pattern observed in wild-type controls. However, the intradermal injection of formalin elicited a significant activation of the putative pain pathway as represented by such areas as the anterior cingulate, somatosensory cortex, parabrachial nucleus, and periaqueductal gray. Notably, comparison of neural responses to capsaicin in wild-type versus knock-out rats uncovered evidence that capsaicin may function in an antinociceptive capacity independent of TRPV1 signaling. Our data suggest that neuroimaging of pain in awake, conscious animals has the potential to inform the neurobiological basis of full and integrated perceptions of pain.

Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in ApcMin/+ mice

OpenAIRE

Chen, Lei; Brar, Manreetpal S.; Leung, Frederick C. C.; Hsiao, W. L. Wendy

2016-01-01

Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetit...

An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

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Melissa S Rotunno

2013-12-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1 protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.

Cardiovascular Benefits of Moderate Exercise Training in Marfan Syndrome: Insights From an Animal Model.

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Mas-Stachurska, Aleksandra; Siegert, Anna-Maria; Batlle, Monsterrat; Gorbenko Del Blanco, Darya; Meirelles, Thayna; Rubies, Cira; Bonorino, Fabio; Serra-Peinado, Carla; Bijnens, Bart; Baudin, Julio; Sitges, Marta; Mont, Lluís; Guasch, Eduard; Egea, Gustavo

2017-09-25

Marfan syndrome (MF) leads to aortic root dilatation and a predisposition to aortic dissection, mitral valve prolapse, and primary and secondary cardiomyopathy. Overall, regular physical exercise is recommended for a healthy lifestyle, but dynamic sports are strongly discouraged in MF patients. Nonetheless, evidence supporting this recommendation is lacking. Therefore, we studied the role of long-term dynamic exercise of moderate intensity on the MF cardiovascular phenotype. In a transgenic mouse model of MF ( Fbn1 C1039G/+ ), 4-month-old wild-type and MF mice were subjected to training on a treadmill for 5 months; sedentary littermates served as controls for each group. Aortic and cardiac remodeling was assessed by echocardiography and histology. The 4-month-old MF mice showed aortic root dilatation, elastic lamina rupture, and tunica media fibrosis, as well as cardiac hypertrophy, left ventricular fibrosis, and intramyocardial vessel remodeling. Over the 5-month experimental period, aortic root dilation rate was significantly greater in the sedentary MF group, compared with the wild-type group (∆mm, 0.27±0.07 versus 0.13±0.02, respectively). Exercise significantly blunted the aortic root dilation rate in MF mice compared with sedentary MF littermates (∆mm, 0.10±0.04 versus 0.27±0.07, respectively). However, these 2 groups were indistinguishable by aortic root stiffness, tunica media fibrosis, and elastic lamina ruptures. In MF mice, exercise also produced cardiac hypertrophy regression without changes in left ventricular fibrosis. Our results in a transgenic mouse model of MF indicate that moderate dynamic exercise mitigates the progression of the MF cardiovascular phenotype. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

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ÁNGELA D ARMENDÁRIZ

2006-01-01

Full Text Available The role of metallothioneins (MT in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-. As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80% belong to two major categories: 1 metabolism; and 2 cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

Automatic Detection of Wild-type Mouse Cranial Sutures

DEFF Research Database (Denmark)

Ólafsdóttir, Hildur; Darvann, Tron Andre; Hermann, Nuno V.

, automatic detection of the cranial sutures becomes important. We have previously built a craniofacial, wild-type mouse atlas from a set of 10 Micro CT scans using a B-spline-based nonrigid registration method by Rueckert et al. Subsequently, all volumes were registered nonrigidly to the atlas. Using......, the observer traced the sutures on each of the mouse volumes as well. The observer outperforms the automatic approach by approximately 0.1 mm. All mice have similar errors while the suture error plots reveal that suture 1 and 2 are cumbersome, both for the observer and the automatic approach. These sutures can...

No evidence for functional inactivation of wild-type p53 protein by MDM2 overexpression in gastric carcinogenesis

NARCIS (Netherlands)

Blok, P.; Craanen, M. E.; Dekker, W.; Offerhaus, G. J.; Tytgat, G. N.

1998-01-01

Inactivation of wild-type p53 during gastric carcinogenesis is usually caused by mutations within exons 5-8 of the p53 gene leading to mutated, usually immunohistochemically detectable p53 proteins. However, functional inactivation of wild-type p53, mimicking mutational inactivation, may also result

Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

DEFF Research Database (Denmark)

Steffensen, Karina Dahl; Waldstrøm, Marianne; Pallisgård, Niels

2013-01-01

OBJECTIVE: The increasing number of negative trials for ovarian cancer treatment has prompted an evaluation of new biologic agents, which in combination with chemotherapy may improve survival. The aim of this study was to investigate the response rate in platinum-resistant, KRAS wild-type ovarian...... cancer patients treated with pegylated liposomal doxorubicin (PLD) supplemented with panitumumab. PATIENTS AND METHODS: Major eligibility criteria were relapsed ovarian/fallopian/peritoneal cancer patients with platinum-resistant disease, measurable disease by GCIG CA125 criteria and KRAS wild-type...

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

Science.gov (United States)

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

2013-01-01

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

Cerebrospinal fluid neurofilament light chain as a biomarker of neurodegeneration in the Tg4510 and MitoPark mouse models

DEFF Research Database (Denmark)

Clement, Amalie; Mitchelmore, Cathy; Andersson, Daniel

2017-01-01

examined whether changes in NF-L levels in brain, plasma, and CSF reflect the changing disease status of preclinical models of neurodegeneration. Using Western Blot and ELISA we characterized NF-L and disease-related proteins in brain, CSF and plasma samples from Tg4510 mice (tauopathy/AD), MitoPark mice...... (PD), and their age-matched control littermates. We found that CSF NF-L clearly discriminates Tg4510 from control littermates, which was not observed for the MitoPark model. However, both Tg4510 and MitoPark showed altered expression and solubilization of NFs compared to control littermates. We found...

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

OpenAIRE

Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.

2007-01-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solution...

Neurofibromin 1 Impairs Natural Killer T-Cell-Dependent Antitumor Immunity against a T-Cell Lymphoma

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Jianyun Liu

2018-01-01

Full Text Available Neurofibromin 1 (NF1 is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/− mice. Nf1+/− mice were found to have similar levels of NKT cells as wildtype (WT littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/− mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/− mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/− mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/− mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

Comparison of the nucleotide sequence of wild-type hepatitis - A virus and its attenuated candidate vaccine derivative

International Nuclear Information System (INIS)

Cohen, J.I.; Rosenblum, B.; Ticehurst, J.R.; Daemer, R.; Feinstone, S.; Purcell, R.H.

1987-01-01

Development of attenuated mutants for use as vaccines is in progress for other viruses, including influenza, rotavirus, varicella-zoster, cytomegalovirus, and hepatitis-A virus (HAV). Attenuated viruses may be derived from naturally occurring mutants that infect human or nonhuman hosts. Alternatively, attenuated mutants may be generated by passage of wild-type virus in cell culture. Production of attenuated viruses in cell culture is a laborious and empiric process. Despite previous empiric successes, understanding the molecular basis for attenuation of vaccine viruses could facilitate future development and use of live-virus vaccines. Comparison of the complete nucleotide sequences of wild-type (virulent) and vaccine (attenuated) viruses has been reported for polioviruses and yellow fever virus. Here, the authors compare the nucleotide sequence of wild-type HAV HM-175 with that of a candidate vaccine derivative

Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone beta1 receptor gene.

Science.gov (United States)

McDonald, M P; Wong, R; Goldstein, G; Weintraub, B; Cheng, S Y; Crawley, J N

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor beta (TRbeta) gene on chromosome 3, representing a mutation of the ligand-binding domain of the TRbeta gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRbeta gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRbeta gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.

Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

Science.gov (United States)

McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD. PMID:10454355

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

International Nuclear Information System (INIS)

Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo

2005-01-01

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F WT ) and attachment (H WT ) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H WT determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F WT reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection

The correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in Surabaya

Directory of Open Access Journals (Sweden)

Puspa Dila Rohmaniar

2017-03-01

Full Text Available Background: Exposure of metals among dental technicians that come from the working environment can lead to the formation reactive oxygen species (ROS. ROS can cause mutations in the p53 gene (p53. The mutation is transversion mutation GuanineThymine. p53 mutations can lead to low expression of the wild-type p53 protein (p53. Wild-type p53 involved in many biological processes such as regulation of genes involved in cell cycle, cell growth after DNA damage, and apoptosis. However, exposure to metals among dental technicians can be prevented through the use of personal protective equipment (PPE during work. Purpose: The purpose of this study was to analyze the correlation between the use of personal protective equipment to wild-type p53 protein levels among dental technicians in Surabaya. Method: This study was observational analytic with cross sectional approach. 40 samples were taken by random sampling. Data were retrieved through interviews and observations. Wild-type p53 was analyzed from saliva with indirect ELISA method. Analysis of data used Kolmogorov Smirnov normality test and a Pearson correlation test. Value significance was p<0.05 (95% confidence level. Result: There was a significant association between the use of personal protective equipment with wild-type p53 levels with p=0.002 Conclusion: The use PPE properly is positively correlated with the wild-type p53 protein levels of dental technicians in Surabaya.

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

Science.gov (United States)

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

Early development and the emergence of individual differences in behavior among littermates of wild rabbit pups.

Science.gov (United States)

Rödel, Heiko G; Bautista, Amando; Roder, Manuel; Gilbert, Caroline; Hudson, Robyn

2017-05-01

the open field, suggesting greater reactivity and responsiveness of relatively lighter pups in these tests. In contrast, relatively heavier pups jumped sooner from the platform, which may have been due to their greater physical maturation. In conclusion, our study shows that individual differences in behavior and associated differences in body temperature and growth are already present during early postnatal life, although such relationships can be easily overlooked, as they predominantly emerge as relative differences among littermates. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Science.gov (United States)

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Comparative Study of Nonautolytic Mutant and Wild-Type Strains of Coprinopsis cinerea Supports an Important Role of Glucanases in Fruiting Body Autolysis.

Science.gov (United States)

Liu, Zhonghua; Niu, Xin; Wang, Jun; Zhang, Wenming; Yang, Mingmei; Liu, Cuicui; Xiong, Yuanjing; Zhao, Yan; Pei, Siyu; Qin, Qin; Zhang, Yu; Yu, Yuan; Yuan, Sheng

2015-11-04

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

Transgenic mice with astrocyte-targeted production of interleukin-6 are resistant to high-fat diet-induced increases in body weight and body fat

DEFF Research Database (Denmark)

Hidalgo, Juan; Florit, Sergi; Giralt, Mercedes

2010-01-01

Interleukin-6 (IL-6) is a major cytokine involved in both normal physiological brain functions and underlying significant neuropathology. IL-6 has been suggested to play a role in the control of body weight but the results are somewhat controversial. In this study we have challenged transgenic mice...... with astrocyte-targeted IL-6 expression (GFAP-IL6 mice) with a high-fat diet (55% kcal from fat) versus a control diet (10%). The results demonstrate that the GFAP-IL6 mice are resistant to high-fat diet-induced increases in body weight and body fat, apparently without altering food intake and with no evidences...... of increased sympathetic tone. The high-fat diet-induced impaired responses to an insulin tolerance test (ITT), and to an oral glucose tolerance test (OGTT) in both genotypes. The GFAP-IL6 mice did not differ from littermate wild-type (WT) mice in ITT, but they were more glucose intolerant following the high...

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...

Blood BDNF concentrations reflect brain-tissue BDNF levels across species

DEFF Research Database (Denmark)

Klein, Anders B; Williamson, Rebecca; Santini, Martin A

2011-01-01

Brain-derived neurotrophic factor (BDNF) is involved in synaptic plasticity, neuronal differentiation and survival of neurons. Observations of decreased serum BDNF levels in patients with neuropsychiatric disorders have highlighted the potential of BDNF as a biomarker, but so far there have been...... no studies directly comparing blood BDNF levels to brain BDNF levels in different species. We examined blood, serum, plasma and brain-tissue BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method. As a control, we included an analysis of blood and brain tissue from...... conditional BDNF knockout mice and their wild-type littermates. Whereas BDNF could readily be measured in rat blood, plasma and brain tissue, it was undetectable in mouse blood. In pigs, whole-blood levels of BDNF could not be measured with a commercially available ELISA kit, but pig plasma BDNF levels (mean...

Blood BDNF concentrations reflect brain-tissue BDNF levels across species

DEFF Research Database (Denmark)

Klein, Anders B; Williamson, Rebecca; Santini, Martin A

2011-01-01

no studies directly comparing blood BDNF levels to brain BDNF levels in different species. We examined blood, serum, plasma and brain-tissue BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method. As a control, we included an analysis of blood and brain tissue from......Brain-derived neurotrophic factor (BDNF) is involved in synaptic plasticity, neuronal differentiation and survival of neurons. Observations of decreased serum BDNF levels in patients with neuropsychiatric disorders have highlighted the potential of BDNF as a biomarker, but so far there have been...... conditional BDNF knockout mice and their wild-type littermates. Whereas BDNF could readily be measured in rat blood, plasma and brain tissue, it was undetectable in mouse blood. In pigs, whole-blood levels of BDNF could not be measured with a commercially available ELISA kit, but pig plasma BDNF levels (mean...

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

Science.gov (United States)

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Variation in photosynthesis and stomatal conductance among red maple (Acer rubrum) urban planted cultivars and wildtype trees in the southeastern United States.

Science.gov (United States)

Lahr, Eleanor C; Dunn, Robert R; Frank, Steven D

2018-01-01

Photosynthesis is a fundamental process that trees perform over fluctuating environmental conditions. This study of red maple (Acer rubrum L.) characterizes photosynthesis, stomatal conductance, and water use efficiency in planted cultivars relative to wildtype trees. Red maple is common in cities, yet there is little understanding of how physiological processes affect the long-term growth, condition, and ecosystem services provided by urban trees. In the first year of our study, we measured leaf-level gas exchange and performed short-term temperature curves on urban planted cultivars and on suburban and rural wildtype trees. In the second year, we compared urban planted cultivars and urban wildtype trees. In the first year, urban planted trees had higher maximum rates of photosynthesis and higher overall rates of photosynthesis and stomatal conductance throughout the summer, relative to suburban or rural wildtype trees. Urban planted trees again had higher maximum rates of photosynthesis in the second year. However, urban wildtype trees had higher water use efficiency as air temperatures increased and similar overall rates of photosynthesis, relative to cultivars, in mid and late summer. Our results show that physiological differences between cultivars and wildtype trees may relate to differences in their genetic background and their responses to local environmental conditions, contingent on the identity of the horticultural variety. Overall, our results suggest that wildtype trees should be considered for some urban locations, and our study is valuable in demonstrating how site type and tree type can inform tree planting strategies and improve long-term urban forest sustainability.

dcc Haploinsufficiency results in blunted sensitivity to cocaine enhancement of reward seeking.

Science.gov (United States)

Reynolds, Lauren M; Gifuni, Anthony J; McCrea, E Tess; Shizgal, Peter; Flores, Cecilia

2016-02-01

Mesocortical dopamine connectivity continues to mature during adolescence. This protracted development confers increased vulnerability for environmental and genetic factors to disrupt mesocortical wiring and subsequently influence responses to drugs of abuse in adulthood. The netrin-1 receptor, DCC, orchestrates medial prefrontal cortex dopamine input during adolescence and dictates the functional organization of local circuitry. Haploinsufficiency of dcc results in increased dopamine innervation to the medial prefrontal cortex, which in turn leads to resilience against the behavioral activating effects of stimulant drugs. However, whether sensitivity to the rewarding effects of drugs of abuse is also altered in dcc haploinsufficiency remains to be resolved. Here, we used the curve-shift method to measure cocaine-induced facilitation of intracranial self-stimulation (ICSS) in adult dcc haploinsufficient mice and wild-type littermates. We found that dcc haploinsufficient mice acquire ICSS behavior at comparable stimulation parameters to wild-type controls. However, cocaine-induced potentiation of ICSS is significantly blunted in dcc haploinsufficient mice. These results are consistent with decreased sensitivity to the rewarding effects of cocaine and/or decreased proclivity to invest effort in the pursuit of reward in dcc haploinsufficient mice. Moreover, these findings suggest that DCC signaling determines adult susceptibility to drug abuse most likely by controlling prefrontal cortex development in adolescence. Copyright © 2015 Elsevier B.V. All rights reserved.

Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells.

Science.gov (United States)

Shi, Ming-Zhu; Xie, De-Yu

2011-04-01

We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

Floral vascular patterns of the double-flowered and wildtype morphs of Nigella damascena L. (Ranunculaceae

Directory of Open Access Journals (Sweden)

Thierry Deroin

2015-05-01

Full Text Available The perianth of the double-flowered morph of Nigella damascena L. consists of spirally inserted petaloid sepals and sepal-like organs, similar in shape and colour to the petaloid sepals of the wild-type flower. It is devoid of petals. We compare the vascularization of each organ category of the double flower with that of the wild-type. We show that the vascular patterns of the sepal-like organs and of the petals are identical, and found an inverse relationship between the number of bracts and the number of sepals in the double-flowered morph. These two surprising findings will influence the future evo-devo studies on this plant model.

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

Science.gov (United States)

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

2015-01-01

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

Inactivation of adipose angiotensinogen reduces adipose tissue macrophages and increases metabolic activity.

Science.gov (United States)

LeMieux, Monique J; Ramalingam, Latha; Mynatt, Randall L; Kalupahana, Nishan S; Kim, Jung Han; Moustaïd-Moussa, Naïma

2016-02-01

The adipose renin-angiotensin system (RAS) has been linked to obesity-induced inflammation, though mechanisms are not completely understood. In this study, adipose-specific angiotensinogen knockout mice (Agt-KO) were generated to determine whether Agt inactivation reduces inflammation and alters the metabolic profile of the Agt-KO mice compared to wild-type (WT) littermates. Adipose tissue-specific Agt-KO mice were created using the Cre-LoxP system with both Agt-KO and WT littermates fed either a low-fat or high-fat diet to assess metabolic changes. White adipose tissue was used for gene/protein expression analyses and WAT stromal vascular cells for metabolic extracellular flux assays. No significant differences were observed in body weight or fat mass between both genotypes on either diet. However, improved glucose clearance was observed in Agt-KO compared to WT littermates, consistent with higher expression of genes involved in insulin signaling, glucose transport, and fatty acid metabolism. Furthermore, Agt inactivation reduced total macrophage infiltration in Agt-KO mice fed both diets. Lastly, stroma vascular cells from Agt-KO mice revealed higher metabolic activity compared to WT mice. These findings indicate that adipose-specific Agt inactivation leads to reduced adipose inflammation and increased glucose tolerance mediated in part via increased metabolic activity of adipose cells. © 2015 The Obesity Society.

RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

Directory of Open Access Journals (Sweden)

Yuanxin Miao

2015-04-01

Full Text Available Myostatin (MSTN, a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph, and zinc metallopeptidase STE24 (Zmpste24. In addition, kyphoscoliosis peptidase (Ky, which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA pathways (Dgki, Dgkz, Plcd4 were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

Science.gov (United States)

Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

2015-04-09

Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

OpenAIRE

Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

2010-01-01

Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the pre...

Aldose Reductase-Deficient Mice Develop Nephrogenic Diabetes Insipidus

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Ho, Horace T. B.; Chung, Sookja K.; Law, Janice W. S.; Ko, Ben C. B.; Tam, Sidney C. F.; Brooks, Heddwen L.; Knepper, Mark A.; Chung, Stephen S. M.

2000-01-01

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus. PMID:10913167

The effect of dietary folic acid deficiency on the cytotoxic and mutagenic responses to methyl methanesulfonate in wild-type and in 3-methyladenine DNA glycosylase-deficient Aag null mice.

Science.gov (United States)

Branda, Richard F; O'Neill, J Patrick; Brooks, Elice M; Powden, Cheryl; Naud, Shelly J; Nicklas, Janice A

2007-02-03

Folic acid deficiency (FA-) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA- diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p=0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA- diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA- wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA- mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA-) had no effect on cytotoxicity, but FA- diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA- diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.

A ten-week biochemistry lab project studying wild-type and mutant bacterial alkaline phosphatase.

Science.gov (United States)

Witherow, D Scott

2016-11-12

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

DEFF Research Database (Denmark)

Ingversen, J.

1975-01-01

Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS......-polyacrylamidegel electrophoresis. Sections through pellets of isolated protein bodies from both the mutant and the wild type revealed protein body structures corresponding with those observed in sections through the intact starchy endosperms. The majority of the wild-type protein bodies was homogeneous spheres accompanied...... that the wild-type protein bodies contained large amounts of prolamines (the storage protein group which is soluble in 55 % isopropanol) and some glutelins (the storage proteins soluble in dilute alkali), whereas the mutant protein bodies have glutelin as the major component and little prolamines...

Comparative behaviour of lab.-cultured and wild-type Dacus oleae flies in the field

International Nuclear Information System (INIS)

Prokopy, R.J.; Haniotakis, G.E.; Economopoulos, A.P.

1975-01-01

Under field conditions, the authors compared the responses of lab.-type (ca. 85 generations under artificial conditions) and wild-type Dacus oleae flies to host plant colour and odour, host fruit colour and shape, small rectangles of different colours and shades, and McPhail-type traps of different colours baited with different odours. Except for the lab.-type flies being relatively more attracted toward red fruit models and small red rectangles and relatively less attracted toward yellow fruit models and small yellow rectangles than the wild type, the qualitative nature of the responses of the two fly types toward the various experimental treatments was essentially the same. Quantitatively, however, consistently smaller percentages of the released lab.-type than the released wild-type flies were recaptured, suggesting that the mobility, flight pattern, or vigour of the two types of flies may be different. (author)

Single-Agent Panitumumab in Frail Elderly Patients With Advanced RAS and BRAF Wild-Type Colorectal Cancer: Challenging Drug Label to Light Up New Hope

Science.gov (United States)

Cremolini, Chiara; Aprile, Giuseppe; Lonardi, Sara; Orlandi, Armando; Mennitto, Alessia; Berenato, Rosa; Antoniotti, Carlotta; Casagrande, Mariaelena; Marsico, Valentina; Marmorino, Federica; Cardellino, Giovanni Gerardo; Bergamo, Francesca; Tomasello, Gianluca; Formica, Vincenzo; Longarini, Raffaella; Giommoni, Elisa; Caporale, Marta; Di Bartolomeo, Maria; Loupakis, Fotios; de Braud, Filippo

2015-01-01

Background. No prospective trials have specifically addressed the efficacy and safety of panitumumab in elderly patients with metastatic colorectal cancer (CRC). We aimed at assessing the efficacy and safety of single agent panitumumab in “frail” elderly patients diagnosed with metastatic RAS and BRAF wild-type CRC. Materials and Methods. Forty elderly patients (aged ≥75 years) with metastatic RAS-BRAF wild-type CRC received off-label prescriptions of single-agent panitumumab at seven Italian institutions. Treatment was administered as first line in patients with absolute contraindication to any chemotherapy or as second-line treatment after failure of a fluoropyrimidine-based treatment, in the presence of contraindication to irinotecan. The outcome measures included objective response rate (ORR), as well as progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and safety. Results. The median PFS and OS were 6.4 months (95% confidence interval [CI]: 4.9–8 months) and 14.3 months (95% CI: 10.9–17.7 months), respectively. ORR was 32.5%, and DCR was 72.5%. Dose reductions related to adverse events (AEs) were reported in 9 (23%) patients, but no permanent treatment discontinuation caused by was reported. The most frequent grade 3 AE was skin rash, with an incidence of 20%. Conclusion. Panitumumab is effective and well-tolerated in frail elderly patients with RAS-BRAF wild-type metastatic CRC and deemed unfit for chemotherapy. A randomized study is needed to confirm these data. Implications for Practice: Treatment of elderly patients with metastatic colorectal cancer represents a difficult challenge in clinical practice. A significant proportion of frail elderly patients do not receive treatment, reflecting ongoing uncertainty of clinical benefit and toxicity of chemotherapy. Unfit condition in this cohort of patients further limits antineoplastic prescription and consequently patient survival. RAS and BRAF wild-type status could

Mid-aged and aged wild-type and progestin receptor knockout (PRKO) mice demonstrate rapid progesterone and 3alpha,5alpha-THP-facilitated lordosis.

Science.gov (United States)

Frye, C A; Sumida, K; Lydon, J P; O'Malley, B W; Pfaff, D W

2006-05-01

Progesterone (P) and its 5alpha-reduced metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), facilitate sexual behavior of rodents via agonist-like actions at intracellular progestin receptors (PRs) and membrane GABA(A)/benzodiazepine receptor complexes (GBRs), respectively. Given that ovarian secretion of progestins declines with aging, whether or not senescent mice are responsive to progestins was of interest. Homozygous PR knockout (PRKO) or wild-type mice that were between 10-12 (mid-aged) or 20-24 (aged) months of age were administered P or 3alpha,5alpha-THP, and the effect on lordosis were examined. Effects of a progestin-priming regimen that enhances PR-mediated (experiment 1) or more rapid, PR-independent effects of progestins (experiments 2 and 3) on sexual behavior were examined. Levels of P, 3alpha,5alpha-THP, and muscimol binding were examined in tissues from aged mice (experiment 4). Wild-type, but not PRKO, mice were responsive when primed with 17beta-estradiol (E(2); 0.5 microg) and administered P (500 microg, subcutaneously). Mid-aged wild-type mice demonstrated greater increases in lordosis 6 h later compared to their pre-P, baseline test than did aged wild-type mice (experiment 1). Lordosis of younger and older wild-type, but not PRKO, mice was significantly increased within 5 min of intravenous (IV) administration of P (100 ng), compared with E(2)-priming alone (experiment 2). However, wild-type and PRKO mice demonstrated significant increases in lordosis 5 min after IV administration of 3alpha,5alpha-THP, an effect which was more pronounced in mid-aged than in aged animals (100 ng-experiment 3). In tissues from aged wild-type and PRKO mice, levels of P, 3alpha,5alpha-THP, and muscimol binding were increased by P administration (experiment 4). PR binding was lower in the cortex of PRKO than that of wild-type mice. Mid-aged and aged PRKO and wild-type mice demonstrated rapid P or 3alpha,5alpha-THP-facilitated lordosis that may be

ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

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The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

Science.gov (United States)

Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

2015-06-01

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

Science.gov (United States)

Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

2016-01-01

Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

Motor coordination defects in mice deficient for the Sam68 RNA-binding protein.

Science.gov (United States)

Lukong, Kiven E; Richard, Stéphane

2008-06-03

The role of RNA-binding proteins in the central nervous system and more specifically their role in motor coordination and learning are poorly understood. We previously reported that ablation of RNA-binding protein Sam68 in mice results in male sterility and delayed mammary gland development and protection against osteoporosis in females. Sam68 however is highly expressed in most regions of the brain especially the cerebellum and thus we investigated the cerebellar-related manifestations in Sam68-null mice. We analyzed the mice for motor function, sensory function, and learning and memory abilities. Herein, we report that Sam68-null mice have motor coordination defects as assessed by beam walking and rotorod performance. Forty-week-old Sam68-null mice (n=12) were compared to their wild-type littermates (n=12). The Sam68-null mice exhibited more hindpaw faults in beam walking tests and fell from the rotating drum at lower speeds and prematurely compared to the wild-type controls. The Sam68-null mice were, however, normal for forelimb strength, tail-hang reflex, balance test, grid walking, the Morris water task, recognition memory, visual discrimination, auditory stimulation and conditional taste aversion. Our findings support a role for Sam68 in the central nervous system in the regulation of motor coordination.

The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

DEFF Research Database (Denmark)

Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

2012-01-01

The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice......)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose...

The Use of EGFR Tyrosine Kinase Inhibitors in EGFR Wild-Type Non-Small-Cell Lung Cancer.

Science.gov (United States)

Stinchcombe, Thomas E

2016-04-01

The objective response rate and progression-free survival observed with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients with metastatic epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) are modest. The adverse events associated with EGFR TKIs are manageable but they must be considered in the context of the limited efficacy. The development of anti-PD-1 immunotherapy as second-line therapy has reduced the role of EGFR TKIs in EGFR wild-type NSCLC. Recently, there has been increased recognition of the benefit of the earlier integration of palliative care and symptom management, and this is reasonable alternative to treatment with an EGFR TKI for many patients. My practice pattern for patients with EGFR wild-type NSCLC is platinum-based chemotherapy as first-line therapy, immunotherapy as second-line therapy, and single-agent chemotherapy as third-line therapy for patients with preserved performance status who want to pursue further therapy. Only a small proportion of patients are eligible for fourth-line therapy, and I prefer to enroll them in clinical trials rather than use EGFR TKIs. I suspect that the use of EGFR TKIs in clinical use and as a comparator arm for clinical trials will continue to decline over the next several years.

Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

Energy Technology Data Exchange (ETDEWEB)

Hartman, P S

1984-01-01

The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation. (orig.). 28 refs.; 4 figs.

Dopamine D₂-Like Receptors and Behavioral Economics of Food Reinforcement.

Science.gov (United States)

Soto, Paul L; Hiranita, Takato; Xu, Ming; Hursh, Steven R; Grandy, David K; Katz, Jonathan L

2016-03-01

Previous studies suggest dopamine (DA) D2-like receptor involvement in the reinforcing effects of food. To determine contributions of the three D2-like receptor subtypes, knockout (KO) mice completely lacking DA D2, D3, or D4 receptors (D2R, D3R, or D4R KO mice) and their wild-type (WT) littermates were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) plotted to assess elasticity of demand. Under both economies, as price increased, food pellets obtained decreased more rapidly (ie, food demand was more elastic) in DA D2R KO mice compared with WT littermates. Extinction of responding was studied in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction. Extinction in DA D2R KO mice occurred less rapidly compared with WT mice in both contexts. Elasticity of food demand was higher in DA D4R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D4R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D3R KO mice and WT littermates. These results indicate that the D2R is the primary DA D2-like receptor subtype mediating the reinforcing effectiveness of food.

Dopamine D2-Like Receptors and Behavioral Economics of Food Reinforcement

Science.gov (United States)

Soto, Paul L; Hiranita, Takato; Xu, Ming; Hursh, Steven R; Grandy, David K; Katz, Jonathan L

2016-01-01

Previous studies suggest dopamine (DA) D2-like receptor involvement in the reinforcing effects of food. To determine contributions of the three D2-like receptor subtypes, knockout (KO) mice completely lacking DA D2, D3, or D4 receptors (D2R, D3R, or D4R KO mice) and their wild-type (WT) littermates were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) plotted to assess elasticity of demand. Under both economies, as price increased, food pellets obtained decreased more rapidly (ie, food demand was more elastic) in DA D2R KO mice compared with WT littermates. Extinction of responding was studied in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction. Extinction in DA D2R KO mice occurred less rapidly compared with WT mice in both contexts. Elasticity of food demand was higher in DA D4R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D4R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D3R KO mice and WT littermates. These results indicate that the D2R is the primary DA D2-like receptor subtype mediating the reinforcing effectiveness of food. PMID:26205210

Mathematical model of the SOS response regulation in wild-type Escherichia coli

International Nuclear Information System (INIS)

Aksenov, S.V.

1997-01-01

Regulation of the SOS response in Escherichia coli, which is a set of inducible cellular reactions introduced after DNA damage, is due to specific interaction of LexA and RecA proteins. LexA protein is a common repressor of the genes of the SOS system, and RecA protein, once transiently activated by the so-called SOS-inducing signal, promotes LexA protein destruction. We have described the SOS regulation by means of differential equations with regard to LexA and RecA concentrations elsewhere. The 'input' function for model equations is the level of the SOS-inducing signal against time. Here we present a means for calculating the concentration of single-stranded DNA (SOS-inducing signal) as a function of time in wild-type cells after ultraviolet irradiation. With model equations one can simulate kinetic curves of SOS regulatory proteins after DNA damage to survey the SOS response kinetics. Simulation of LexA protein kinetics agrees with experimental data. We compare simulated LexA kinetic curves in wild-type and uνr - mutant bacteria, which is useful in investigating the way uνrABC-dependent excision repair modulates the SOS response kinetics. Possible applications of the model to investigating various aspects of the SOS induction are discussed

MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

Science.gov (United States)

Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

2016-10-01

Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (PMLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas can harbor KRAS mutations and arise from precursor polyps resembling conventional tubular/tubulovillous adenomas.

Effects of stimulus salience on touchscreen serial reversal learning in a mouse model of fragile X syndrome

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Dickson, Price E.; Corkill, Beau; McKimm, Eric; Miller, Mellessa M.; Calton, Michele A.; Goldowitz, Daniel; Blaha, Charles D.; Mittleman, Guy

2013-01-01

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability in males and the most common genetic cause of autism. Although executive dysfunction is consistently found in humans with FXS, evidence of executive dysfunction in Fmr1 KO mice, a mouse model of FXS, has been inconsistent. One possible explanation for this is that executive dysfunction in Fmr1 KO mice, similar to humans with FXS, is only evident when cognitive demands are high. Using touchscreen operant conditioning chambers, male Fmr1 KO mice and their male wildtype littermates were tested on the acquisition of a pairwise visual discrimination followed by four serial reversals of the response rule. We assessed reversal learning performance under two different conditions. In the first, the correct stimulus was salient and the incorrect stimulus was non-salient. In the second and more challenging condition, the incorrect stimulus was salient and the correct stimulus was non-salient; this increased cognitive load by introducing conflict between sensory-driven (i.e., bottom-up) and task-dependent (i.e., top-down) signals. Fmr1 KOs displayed two distinct impairments relative to wildtype littermates. First, Fmr1 KOs committed significantly more learning-type errors during the second reversal stage, but only under high cognitive load. Second, during the first reversal stage, Fmr1 KOs committed significantly more attempts to collect a reward during the timeout following an incorrect response. These findings indicate that Fmr1 KO mice display executive dysfunction that, in some cases, is only evident under high cognitive load. PMID:23747611

Prohibitin-induced obesity leads to anovulation and polycystic ovary in mice

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Sudharsana Rao Ande

2017-06-01

Full Text Available Polycystic ovary syndrome (PCOS is a prevalent endocrine disorder and the most common cause of female infertility. However, its etiology and underlying mechanisms remain unclear. Here we report that a transgenic obese mouse (Mito-Ob developed by overexpressing prohibitin in adipocytes develops polycystic ovaries. Initially, the female Mito-Ob mice were equally fertile to their wild-type littermates. The Mito-Ob mice began to gain weight after puberty, became significantly obese between 3-6 months of age, and ∼25% of them had become infertile by 9 months of age. Despite obesity, female Mito-Ob mice maintained glucose homeostasis and insulin sensitivity similar to their wild-type littermates. Mito-Ob mice showed morphologically distinct polycystic ovaries and elevated estradiol, but normal testosterone and insulin levels. Histological analysis of the ovaries showed signs of impaired follicular dynamics, such as preantral follicular arrest and reduced number, or absence, of corpus luteum. The ovaries of the infertile Mito-Ob mice were closely surrounded by periovarian adipose tissue, suggesting a potential role in anovulation. Collectively, these data suggest that elevated estradiol and obesity per se might lead to anovulation and polycystic ovaries independent of hyperinsulinemia and hyperandrogenism. As obesity often coexists with other abnormalities known to be involved in the development of PCOS such as insulin resistance, compensatory hyperinsulinemia and hyperandrogenism, the precise role of these factors in PCOS remains unclear. Mito-Ob mice provide an opportunity to study the effects of obesity on anovulation and ovarian cyst formation independent of the major drivers of obesity-linked PCOS.

Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone

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Bikle, Daniel D.; Sakata, Takeshi; Leary, Colin; Elalieh, Hashem; Ginzinger, David; Rosen, Clifford J.; Beamer, Wesley; Majumdar, Sharmila; Halloran, Bernard P.

2002-01-01

Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin-like growth factor (IGF) I production, but it remains uncertain whether IGF-I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF-I-deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 microg/kg) or vehicle for 2 weeks after which their tibias were examined for fat-free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild-type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF-I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several-fold more in response to PTH than the mRNAs in the bones from their wild-type littermates. These results indicate that IGF-I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.

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

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

2015-07-10

Interleukin 1 (IL-1) plays a critical role in stress responses, and its mRNA is induced in the brain by restraint stress. Previously, we reported that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lacked IL-1Ra molecules that antagonize the IL-1 receptor, showed anti-depression-like behavior via adrenergic modulation at the age of 8 weeks. Here, we report that IL-1Ra KO mice display an anxiety-like phenotype that is induced spontaneously by aging in the elevated plus-maze (EPM) test. This anxiety-like phenotype was improved by the administration of diazepam. The expression of the anxiety-related molecule glucocorticoid receptor (GR) was significantly reduced in 20-week-old but not in 11-week-old IL-1Ra KO mice compared to wild-type (WT) littermates. The expression of the mineralocorticoid receptor (MR) was not altered between IL-1Ra KO mice and WT littermates at either 11 or 20 weeks old. Analysis of monoamine concentration in the hippocampus revealed that tryptophan, the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA), and the dopamine metabolite homovanillic acid (HVA) were significantly increased in 20-week-old IL-1Ra KO mice compared to littermate WT mice. These findings strongly suggest that the anxiety-like behavior observed in older mice was caused by the complicated alteration of monoamine metabolism and/or GR expression in the hippocampus. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Craniofacial Statistical Deformation Models of Wild-type mice and Crouzon mice

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Ólafsdóttir, Hildur; Darvann, Tron Andre; Ersbøll, Bjarne Kjær

2007-01-01

Crouzon syndrome is characterised by the premature fusion of cranial sutures and synchondroses leading to craniofacial growth disturbances. The gene causing the syndrome was discovered approximately a decade ago and recently the first mouse model of the syndrome was generated. In this study, a set...... of Micro CT scannings of the heads of wild-type (normal) mice and Crouzon mice were investigated. Statistical deformation models were built to assess the anatomical differences between the groups, as well as the within-group anatomical variation. Following the approach by Rueckert et al. we built an atlas...

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

DEFF Research Database (Denmark)

Maccarana, M.; Kalamajski, S.; Kongsgaard, M.

2009-01-01

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks......-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found...... that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile...

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and nontransformed tomato roots of either wild-type or AM-defective phenotypes in monoxenic cultures.

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Bago, Alberto; Cano, Custodia; Toussaint, Jean-Patrick; Smith, Sally; Dickson, Sandy

2006-09-01

Monoxenic symbioses between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and two nontransformed tomato root organ cultures (ROCs) were established. Wild-type tomato ROC from cultivar "RioGrande 76R" was employed as a control for mycorrhizal colonization and compared with its mutant line (rmc), which exhibits a highly reduced mycorrhizal colonization (rmc) phenotype. Structural features of the two root lines were similar when grown either in soil or under in vitro conditions, indicating that neither monoxenic culturing nor the rmc mutation affected root development or behavior. Colonization by G. intraradices in monoxenic culture of the wild-type line was low (<10%) but supported extensive development of extraradical mycelium, branched absorbing structures, and spores. The reduced colonization of rmc under monoxenic conditions (0.6%) was similar to that observed previously in soil. Extraradical development of runner hyphae was low and proportional to internal colonization. Few spores were produced. These results might suggest that carbon transfer may be modified in the rmc mutant. Our results support the usefulness of monoxenically obtained mycorrhizas for investigation of AM colonization and intraradical symbiotic functioning.

Mild myopathy is associated with COMP but not MATN3 mutations in mouse models of genetic skeletal diseases.

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Katarzyna A Piróg

Full Text Available Pseudoachondroplasia (PSACH and multiple epiphyseal dysplasia (MED are skeletal disorders resulting from mutations in COMP, matrilin-3 or collagen IX and are characterised by short-limbed dwarfism and premature osteoarthritis. Interestingly, recent reports suggest patients can also manifest with muscle weakness. Here we present a detailed analysis of two mouse models of the PSACH/MED disease spectrum; ΔD469 T3-COMP (PSACH and V194D matrilin-3 (MED. In grip test experiments T3-COMP mice were weaker than wild-type littermates, whereas V194D mice behaved as controls, confirming that short-limbed dwarfism alone does not contribute to PSACH/MED-related muscle weakness. Muscles from T3-COMP mice showed an increase in centronuclear fibers at the myotendinous junction. T3-COMP tendons became more lax in cyclic testing and showed thicker collagen fibers when compared with wild-type tissue; matrilin-3 mutant tissues were indistinguishable from controls. This comprehensive study of the myopathy associated with PSACH/MED mutations enables a better understanding of the disease progression, confirms that it is genotype specific and that the limb weakness originates from muscle and tendon pathology rather than short-limbed dwarfism itself. Since some patients are primarily diagnosed with neuromuscular symptoms, this study will facilitate better awareness of the differential diagnoses that might be associated with the PSACH/MED spectrum and subsequent care of PSACH/MED patients.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

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Cui Shang-jin

2010-05-01

Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

Science.gov (United States)

2010-01-01

A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance. PMID:20433759

Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice

OpenAIRE

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

2015-01-01

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

PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle

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Leick, Lotte; Wojtaszewski, Jørgen F P; Johansen, Sune T.

2008-01-01

The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor-gamma coactivator (PGC) 1alpha is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1alpha knockout (KO) and littermate wild-type (WT) mice performed....... Resting muscles of the PGC-1alpha KO mice had lower ( approximately 20%) cytochrome c (cyt c), cytochrome oxidase (COX) I, and aminolevulinate synthase (ALAS) 1 mRNA and protein levels than WT, but similar levels of AMP-activated protein kinase (AMPK) alpha1, AMPKalpha2, and hexokinase (HK) II compared...

The L-alpha-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in L-arginine-induced insulin release

DEFF Research Database (Denmark)

Smajilovic, Sanela; Clemmensen, Christoffer; Johansen, Lars Dan

2013-01-01

insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by L-arginine did not stimulate insulin secretion. We also investigated central...... metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in L-arginine-induced insulin release and glucose...

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

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

2016-01-01

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

Phosphorylated EGFR expression may predict outcome of EGFR-TKIs therapy for the advanced NSCLC patients with wild-type EGFR

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Wang Fen

2012-08-01

Full Text Available Abstract Background EGFR mutation is a strong predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with EGFR wild-type are responsive to TKIs, suggesting that other determinants of outcome besides EGFR mutation might exist. We hypothesized that activation of phosphorylated EGFR could be a potential predictive biomarker to EGFR-TKIs treatment among patients in wild-type EGFR. Method Total of 205 stage IIIb and IV NSCLC patients, tissue samples of whom were available for molecular analysis, were enrolled in this study. The phosphorylation of EGFR at tyrosine 1068 (pTyr1068 and 1173 (pTyr1173 were assessed by immunohistochemistry, and EGFR mutations were detected by denaturing high performance liquid chromatograph (DHPLC. Results Among 205 patients assessable for EGFR mutation and phosphorylation analysis, 92 (44.9% were EGFR mutant and 165 patients (57.6% had pTyr1173 expression. Superior progression-free survival (PFS was seen after EGFR-TKIs therapy in patients with pTyr1068 expression compared to pTyr1068 negative ones (median PFS 7.0 months vs. 1.2 months, P P = 0.016. In subgroup of patients with wild-type EGFR, pTyr1068 expression positive ones had a significantly prolonged PFS (4.2 months vs.1.2 months P  Conclusion pTyr1068 may be a predictive biomarker for screening the population for clinical response to EGFR-TKIs treatment; especially for patients with wild-type EGFR.

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

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Jodi L McGill

Full Text Available Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

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Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

Inhibition of colony-stimulating factor 1 receptor early in disease ameliorates motor deficits in SCA1 mice.

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Qu, Wenhui; Johnson, Andrea; Kim, Joo Hyun; Lukowicz, Abigail; Svedberg, Daniel; Cvetanovic, Marija

2017-05-25

Polyglutamine (polyQ) expansion in the protein Ataxin-1 (ATXN1) causes spinocerebellar ataxia type 1 (SCA1), a fatal dominantly inherited neurodegenerative disease characterized by motor deficits, cerebellar neurodegeneration, and gliosis. Currently, there are no treatments available to delay or ameliorate SCA1. We have examined the effect of depleting microglia during the early stage of disease by using PLX, an inhibitor of colony-stimulating factor 1 receptor (CSFR1), on disease severity in a mouse model of SCA1. Transgenic mouse model of SCA1, ATXN1[82Q] mice, and wild-type littermate controls were treated with PLX from 3 weeks of age. The effects of PLX on microglial density, astrogliosis, motor behavior, atrophy, and gene expression of Purkinje neurons were examined at 3 months of age. PLX treatment resulted in the elimination of 70-80% of microglia from the cerebellum of both wild-type and ATXN1[82Q] mice. Importantly, PLX ameliorated motor deficits in SCA1 mice. While we have not observed significant improvement in the atrophy or disease-associated gene expression changes in Purkinje neurons upon PLX treatment, we have detected reduced expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) and increase in the protein levels of wild-type ataxin-1 and post-synaptic density protein 95 (PSD95) that may help improve PN function. A decrease in the number of microglia during an early stage of disease resulted in the amelioration of motor deficits in SCA1 mice.

Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice

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Ugen Kenneth E

2004-06-01

Full Text Available Abstract Background In prior work we detected reduced anti-Aβ antibody titers in Aβ-vaccinated transgenic mice expressing the human amyloid precursor protein (APP compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Aβ peptide, an Aβ peptide containing the "Dutch Mutation", E22Q, or a wild-type Aβ peptide conjugated to papillomavirus virus-like particles (VLPs. Results Anti-Aβ antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Aβ E22Q. One concern was that human Aβ derived from the APP transgene might mask anti-Aβ antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Aβ antibody titers 20–40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Aβ titers were still lower in transgenic mice vaccinated with wild-type Aβ or E22Q Aβ relative to non-transgenic mice. Importantly, the dissociated anti-Aβ titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Aβ back to the dissociated serum blocked the increase in antibody titers. Conclusions Circulating human Aβ can interfere with ELISA assay measurements of anti-Aβ titers. The E22Q Aβ peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Aβ abrogate the effects of Aβ self-tolerance.

Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

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Teemu Aitta-aho

Full Text Available In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test; in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards, in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009

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Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J.; Rota, Paul A.; Xu, Wenbo

2013-01-01

Background China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Principal Findings Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was measles in China. PMID:24073194

Genome Sequences of Three Vaccine Strains and Two Wild-Type Canine Distemper Virus Strains from a Recent Disease Outbreak in South Africa.

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Loots, Angelika K; Du Plessis, Morné; Dalton, Desiré Lee; Mitchell, Emily; Venter, Estelle H

2017-07-06

Canine distemper virus causes global multihost infectious disease. This report details complete genome sequences of three vaccine and two new wild-type strains. The wild-type strains belong to the South African lineage, and all three vaccine strains to the America 1 lineage. This constitutes the first genomic sequences of this virus from South Africa. Copyright © 2017 Loots et al.

Cognitive assessment of mice strains heterozygous for cell-adhesion genes reveals strain-specific alterations in timing.

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Gallistel, C R; Tucci, Valter; Nolan, Patrick M; Schachner, Melitta; Jakovcevski, Igor; Kheifets, Aaron; Barboza, Luendro

2014-03-05

We used a fully automated system for the behavioural measurement of physiologically meaningful properties of basic mechanisms of cognition to test two strains of heterozygous mutant mice, Bfc (batface) and L1, and their wild-type littermate controls. Both of the target genes are involved in the establishment and maintenance of synapses. We find that the Bfc heterozygotes show reduced precision in their representation of interval duration, whereas the L1 heterozygotes show increased precision. These effects are functionally specific, because many other measures made on the same mice are unaffected, namely: the accuracy of matching temporal investment ratios to income ratios in a matching protocol, the rate of instrumental and classical conditioning, the latency to initiate a cued instrumental response, the trials on task and the impulsivity in a switch paradigm, the accuracy with which mice adjust timed switches to changes in the temporal constraints, the days to acquisition, and mean onset time and onset variability in the circadian anticipation of food availability.

Cylindromatosis mediates neuronal cell death in vitro and in vivo.

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Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

2018-01-19

The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

DEFF Research Database (Denmark)

Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus

2011-01-01

Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...... this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs...... of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis...

Matrix metalloproteinase-2 plays a critical role in overload induced skeletal muscle hypertrophy.

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Zhang, Qia; Joshi, Sunil K; Lovett, David H; Zhang, Bryon; Bodine, Sue; Kim, Hubert T; Liu, Xuhui

2014-01-01

extracellular matrix (ECM) components are instrumental in maintaining homeostasis and muscle fiber functional integrity. Skeletal muscle hypertrophy is associated with ECM remodeling. Specifically, recent studies have reported the involvement of matrix metalloproteinases (MMPs) in muscle ECM remodeling. However, the functional role of MMPs in muscle hypertrophy remains largely unknown. in this study, we examined the role of MMP-2 in skeletal muscle hypertrophy using a previously validated method where the plantaris muscle of mice were subjected to mechanical overload due to the surgical removal of synergist muscles (gastrocnemius and soleus). following two weeks of overload, we observed a significant increase in MMP-2 activity and up-regulation of ECM components and remodeling enzymes in the plantaris muscles of wild-type mice. However, MMP-2 knockout mice developed significantly less hypertrophy and ECM remodeling in response to overload compared to their wild-type littermates. Investigation of protein synthesis rate and Akt/mTOR signaling revealed no difference between wild-type and MMP-2 knockout mice, suggesting that a difference in hypertrophy was independent of protein synthesis. taken together, our results suggest that MMP-2 is a key mediator of ECM remodeling in the setting of skeletal muscle hypertrophy.

Maintenance erlotinib in advanced nonsmall cell lung cancer: cost-effectiveness in EGFR wild-type across Europe

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Walleser S

2012-09-01

Full Text Available Silke Walleser,1 Joshua Ray,2 Helge Bischoff,3 Alain Vergnenègre,4 Hubertus Rosery,5 Christos Chouaid,6 David Heigener,7 Javier de Castro Carpeño,8 Marcello Tiseo,9 Stefan Walzer21Health Economic Consultancy, Renens, Switzerland; 2F Hoffmann-La Roche Pharmaceuticals AG, Basel, Switzerland; 3Thoracic Hospital of Heidelberg, Heidelberg, Germany; 4Limoges University Hospital, Limoges, France; 5Assessment-in-Medicine GmbH, Loerrach, Germany; 6Hospital Saint Antoine, Paris, France; 7Hospital Grosshansdorf, Grosshansdorf, Germany; 8University Hospital La Paz, Madrid, Spain; 9University Hospital of Parma, Parma, ItalyBackground: First-line maintenance erlotinib in patients with locally advanced or metastatic nonsmall cell lung cancer (NSCLC has demonstrated significant overall survival and progression-free survival benefits compared with best supportive care plus placebo, irrespective of epidermal growth factor receptor (EGFR status (SATURN trial. The cost-effectiveness of first-line maintenance erlotinib in the overall SATURN population has been assessed and published recently, but analyses according to EGFR mutation status have not been performed yet, which was the rationale for assessing the cost-effectiveness of first-line maintenance erlotinib specifically in EGFR wild-type metastatic NSCLC.Methods: The incremental cost per life-year gained of first-line maintenance erlotinib compared with best supportive care in patients with EGFR wild-type stable metastatic NSCLC was assessed for five European countries (the United Kingdom, Germany, France, Spain, and Italy with an area-under-the-curve model consisting of three health states (progression-free survival, progressive disease, death. Log-logistic survival functions were fitted to Phase III patient-level data (SATURN to model progression-free survival and overall survival. The first-line maintenance erlotinib therapy cost (modeled for time to treatment cessation, medication cost in later lines, and

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer.

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Jatin Roper

Full Text Available To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC.PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC(50 = 9.0-14.3 nM. Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01 vs. a 43% decrease (p = 0.008 in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003, no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013.These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

Proteomics of Arabidopsis Seed Germination : a Comparative Study of Wild-Type and Gibberellin-Deficient Seeds

NARCIS (Netherlands)

Gallardo, K.; Job, C.; Groot, S.P.C.; Puype, M.; Vandekerckhove, J.; Job, D.

2002-01-01

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA

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

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Andrew W Walker

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

Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

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Ming Liu

2010-10-01

Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

Tumour gene expression predicts response to cetuximab in patients with KRAS wild-type metastatic colorectal cancer.

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Baker, J B; Dutta, D; Watson, D; Maddala, T; Munneke, B M; Shak, S; Rowinsky, E K; Xu, L-A; Harbison, C T; Clark, E A; Mauro, D J; Khambata-Ford, S

2011-02-01

Although it is accepted that metastatic colorectal cancers (mCRCs) that carry activating mutations in KRAS are unresponsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, a significant fraction of KRAS wild-type (wt) mCRCs are also unresponsive to anti-EGFR therapy. Genes encoding EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are promising gene expression-based markers but have not been incorporated into a test to dichotomise KRAS wt mCRC patients with respect to sensitivity to anti-EGFR treatment. We used RT-PCR to test 110 candidate gene expression markers in primary tumours from 144 KRAS wt mCRC patients who received monotherapy with the anti-EGFR antibody cetuximab. Results were correlated with multiple clinical endpoints: disease control, objective response, and progression-free survival (PFS). Expression of many of the tested candidate genes, including EREG and AREG, strongly associate with all clinical endpoints. Using multivariate analysis with two-layer five-fold cross-validation, we constructed a four-gene predictive classifier. Strikingly, patients below the classifier cutpoint had PFS and disease control rates similar to those of patients with KRAS mutant mCRC. Gene expression appears to identify KRAS wt mCRC patients who receive little benefit from cetuximab. It will be important to test this model in an independent validation study.

Evaluation of MIC Strip Isavuconazole test for susceptibility testing of wild-type and non-wild-type Aspergillus fumigatus isolates

DEFF Research Database (Denmark)

Arendrup, Maiken Cavling; Verweij, Paul; Nielsen, Henrik Vedel

2017-01-01

We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essent......We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2...

CD1d-dependent NKT cells play a protective role in acute and chronic arthritis models by ameliorating antigen-specific Th1 responses

DEFF Research Database (Denmark)

Teige, Anna; Bockermann, Robert; Hasan, Maruf

2010-01-01

-induced arthritis (AIA) and collagen-induced arthritis (CIA), to evaluate acute and chronic arthritis in CD1d knockout mice and mice depleted of NK1.1(+) cells. CD1d-deficient mice developed more severe AIA compared with wild-type littermates, with a higher degree of inflammation and proteoglycan depletion. Chronic...... arthritis in CIA was also worse in the absence of CD1d-dependent NKTs. Elevated levels of Ag-specific IFN-gamma production accompanied these findings rather than changes in IL-17alpha. Depletion of NK1.1(+) cells supported these findings in AIA and CIA. This report provides support for CD1d-dependent NKTs...

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

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Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

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Ya-Ling Yang

2017-01-01

Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Involvement of Amylin and Leptin in the Development of Projections from the Area Postrema to the Nucleus of the Solitary Tract

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Kathrin Abegg

2017-11-01

Full Text Available The area postrema (AP and the nucleus of the solitary tract (NTS are important hindbrain centers involved in the control of energy homeostasis. The AP mediates the anorectic action and the inhibitory effect on gastric emptying induced by the pancreatic hormone amylin. Amylin’s target cells in the AP project to the NTS, an integrative relay center for enteroceptive signals. Perinatal hormonal and metabolic factors influence brain development. A postnatal surge of the adipocyte-derived hormone leptin represents a developmental signal for the maturation of projections between hypothalamic nuclei controlling energy balance. Amylin appears to promote neurogenesis in the AP in adult rats. Here, we examined whether amylin and leptin are required for the development of projections from the AP to the NTS in postnatal and adult mice by conducting neuronal tracing studies with DiI in amylin- (IAPP−/− and leptin-deficient (ob/ob mice. Compared to wild-type littermates, postnatal (P10 and adult (P60 IAPP−/− mice showed a significantly reduced density of AP-NTS projections. While AP projections were also reduced in postnatal (P14 ob/ob mice, AP-NTS fiber density did not differ between adult ob/ob and wild-type animals. Our findings suggest a crucial function of amylin for the maturation of neuronal brainstem pathways controlling energy balance and gastrointestinal function. The impaired postnatal development of neuronal AP-NTS projections in ob/ob mice appears to be compensated in this experimental model during later brain maturation. It remains to be elucidated whether an amylin- and leptin-dependent modulation in neuronal development translates into altered AP/NTS-mediated functions.

Fully-Automated μMRI Morphometric Phenotyping of the Tc1 Mouse Model of Down Syndrome.

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Nick M Powell

Full Text Available We describe a fully automated pipeline for the morphometric phenotyping of mouse brains from μMRI data, and show its application to the Tc1 mouse model of Down syndrome, to identify new morphological phenotypes in the brain of this first transchromosomic animal carrying human chromosome 21. We incorporate an accessible approach for simultaneously scanning multiple ex vivo brains, requiring only a 3D-printed brain holder, and novel image processing steps for their separation and orientation. We employ clinically established multi-atlas techniques-superior to single-atlas methods-together with publicly-available atlas databases for automatic skull-stripping and tissue segmentation, providing high-quality, subject-specific tissue maps. We follow these steps with group-wise registration, structural parcellation and both Voxel- and Tensor-Based Morphometry-advantageous for their ability to highlight morphological differences without the laborious delineation of regions of interest. We show the application of freely available open-source software developed for clinical MRI analysis to mouse brain data: NiftySeg for segmentation and NiftyReg for registration, and discuss atlases and parameters suitable for the preclinical paradigm. We used this pipeline to compare 29 Tc1 brains with 26 wild-type littermate controls, imaged ex vivo at 9.4T. We show an unexpected increase in Tc1 total intracranial volume and, controlling for this, local volume and grey matter density reductions in the Tc1 brain compared to the wild-types, most prominently in the cerebellum, in agreement with human DS and previous histological findings.

Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

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Jakobsen, Oyvind M; Brautaset, Trygve; Degnes, Kristin F; Heggeset, Tonje M B; Balzer, Simone; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2009-02-01

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

Expression of oxidative phosphorylation components in mitochondria of long-living Ames dwarf mice.

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Brown-Borg, Holly M; Johnson, W Thomas; Rakoczy, Sharlene G

2012-02-01

Reduced signaling of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) pathway is associated with extended life span in several species. Ames dwarf mice are GH-deficient and live >50% longer than wild-type littermates. Previously, we have shown that tissues from Ames mice exhibit elevated levels of antioxidative enzymes, less H(2)O(2) production, and lower oxidative damage suggesting that mitochondrial function may differ between genotypes. To explore the relationship between hormone deficiency and mitochondria in mice with extended longevity, we evaluated activity, protein, and gene expression of oxidative phosphorylation components in dwarf and wild-type mice at varying ages. Liver complex I + III activity was higher in dwarf mice compared to wild-type mice. The activity of I + III decreased between 3 and 20 months of age in both genotypes with greater declines in wild-type mice in liver and skeletal muscle. Complex IV activities in the kidney were elevated in 3- and 20-month-old dwarf mice relative to wild-type mice. In Ames mice, protein levels of the 39 kDa complex I subunit were elevated at 20 months of age when compared to wild-type mouse mitochondria for every tissue examined. Kidney and liver mitochondria from 20-month-old dwarf mice had elevated levels of both mitochondrially-encoded and nuclear-encoded complex IV proteins compared to wild-type mice (p dwarf mice. Overall, we found that several components of the oxidative phosphorylation (OXPHOS) system were elevated in Ames mice. Mitochondrial to nuclear DNA ratios were not different between genotypes despite the marked increase in PGC-1α levels in dwarf mice. The increased OXPHOS activities, along with lower ROS production in dwarf mice, predict enhanced mitochondrial function and efficiency, two factors likely contributing to long-life in Ames mice.

Overexpression of human SOD1 improves survival of mice susceptible to endotoxic shock

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Charchaflieh J

2012-07-01

Full Text Available Jean Charchaflieh,1,2 Georges I Labaze,1 Pulsar Li,1 Holly Van Remmen,3 Haekyung Lee,1 Helen Stutz,1 Arlan Richardson,3 Asher Emanuel,1 Ming Zhang1,41Department of Anesthesiology, State University of New York (SUNY Downstate Medical Center, New York, NY, USA; 2Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA; 3Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 4Department of Cell Biology, State University of New York (SUNY Downstate Medical Center, New York, NY, USABackground: Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1 against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.Methods: Transgenic (tg mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1 tg mice with mixed genetic background (tg-JAX; (2 wild-type (WT littermates of tg-JAX strain (WT-JAX; (3 tg mice with C57BL/6J background (tg-TX; (4 WT littermates of tg-TX strain (WT-TX. Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval were analyzed using Kaplan–Meier survival curves.Results: Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX compared with their WT littermates (WT-JAX and WT-TX, respectively. Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX, but such aberrant changes were not

Altered Baseline and Nicotine-Mediated Behavioral and Cholinergic Profiles in ChAT-Cre Mouse Lines.

Science.gov (United States)

Chen, Edison; Lallai, Valeria; Sherafat, Yasmine; Grimes, Nickolas P; Pushkin, Anna N; Fowler, J P; Fowler, Christie D

2018-02-28

The recent development of transgenic rodent lines expressing cre recombinase in a cell-specific manner, along with advances in engineered viral vectors, has permitted in-depth investigations into circuit function. However, emerging evidence has begun to suggest that genetic modifications may introduce unexpected caveats. In the current studies, we sought to extensively characterize male and female mice from both the ChAT (BAC) -Cre mouse line, created with the bacterial artificial chromosome (BAC) method, and ChAT (IRES) -Cre mouse line, generated with the internal ribosome entry site (IRES) method. ChAT (BAC) -Cre transgenic and wild-type mice did not differ in general locomotor behavior, anxiety measures, drug-induced cataplexy, nicotine-mediated hypolocomotion, or operant food training. However, ChAT (BAC) -Cre transgenic mice did exhibit significant deficits in intravenous nicotine self-administration, which paralleled an increase in vesicular acetylcholine transporter and choline acetyltransferase (ChAT) hippocampal expression. For the ChAT (IRES) -Cre line, transgenic mice exhibited deficits in baseline locomotor, nicotine-mediated hypolocomotion, and operant food training compared with wild-type and hemizygous littermates. No differences among ChAT (IRES) -Cre wild-type, hemizygous, and transgenic littermates were found in anxiety measures, drug-induced cataplexy, and nicotine self-administration. Given that increased cre expression was present in the ChAT (IRES) -Cre transgenic mice, as well as a decrease in ChAT expression in the hippocampus, altered neuronal function may underlie behavioral phenotypes. In contrast, ChAT (IRES) -Cre hemizygous mice were more similar to wild-type mice in both protein expression and the majority of behavioral assessments. As such, interpretation of data derived from ChAT-Cre rodents must consider potential limitations dependent on the line and/or genotype used in research investigations. SIGNIFICANCE STATEMENT Altered

CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation

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Drexler Hans G

2010-04-01

Full Text Available Abstract Background CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML, an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPα is a negative regulator of CD7 and which other regulatory mechanisms might be involved. Results As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines. Conclusion We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

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Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

2010-05-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Effects of social stress and intrauterine position on sexual phenotype in wild-type house mice (Mus musculus)

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William J. Zielinski; John G. Vandenbergh; Monica M. Montano

1991-01-01

Wild-type house mice were used to test the effect of intrauterine position on anogenital distance (AGD) and to verify whether crowding stress would masculinize female pups, developing at all intrauterine positions, as has been demonstrated in CF-1 mice stressed by restraint, heat, and...

Effect of running exercise on the number of the neurons in the hippocampus of young transgenic APP/PS1 mice.

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Jiang, Lin; Ma, Jing; Zhang, Yi; Zhou, Chun-Ni; Zhang, Lei; Chao, Feng-Lei; Chen, Lin-Mu; Jiang, Rong; Wu, Hong; Tang, Yong

2018-08-01

To investigate the effect of running exercise on the number of the neurons in the hippocampus of young APP/PS1 mice, twenty 6-month-old male APP/ PS1 transgenic mice were randomly divided into the APP/PS1 control (AD control) group and the APP/PS1 running (AD running) group (10 mice per group), and ten wild-type mice of the littermate were regarded as the wild-type (WT) group. The AD running mice ran on motorized treadmill machiene for 4 months, while the WT mice and AD control mice were housed in standard condition without running. Then, Morris water maze tests (MWM) were used to assess the special learning and memory abilities of mice in three groups. The stereological methods were used to quantitatively evaluate the volume of the hippocampus, CA1/2, CA3 and the dentate gyrus (DG) and count the number of the neurons in CA1/2, CA3 and DG. We found that 4-month running effectively shortened the escape latency of young APP/PS1 control mice in MWM. More importantly, 4-month running effectively increased the volumes of the hippocampus, CA1/2, CA3 and DG and increased the number of neurons in CA1/2, CA3 and DG in young APP/PS1 mice. The present results suggested that 4-month running has significant beneficial effects on the spatial learning and memory capacities of young APP/PS1 mice and could delay the progress of atrophy of hippocampus and the neuron death in CA1/2, CA3 and DG in young APP/PS1 mice. Copyright © 2018 Elsevier B.V. All rights reserved.

Transgenic mice expressing constitutive active MAPKAPK5 display gender-dependent differences in exploration and activity

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Moens Ugo

2007-11-01

Full Text Available Abstract Background The mitogen-activated protein kinases, MAPKs for short, constitute cascades of signalling pathways involved in the regulation of several cellular processes that include cell proliferation, differentiation and motility. They also intervene in neurological processes like fear conditioning and memory. Since little remains known about the MAPK-Activated Protein Kinase, MAPKAPK5, we constructed the first MAPKAPK knockin mouse model, using a constitutive active variant of MAPKAPK5 and analyzed the resulting mice for changes in anxiety-related behaviour. Methods We performed primary SHIRPA observations during background breeding into the C57BL/6 background and assessed the behaviour of the background-bred animals on the elevated plus maze and in the light-dark test. Our results were analyzed using Chi-square tests and homo- and heteroscedatic T-tests. Results Female transgenic mice displayed increased amounts of head dips and open arm time on the maze, compared to littermate controls. In addition, they also explored further into the open arm on the elevated plus maze and were less active in the closed arm compared to littermate controls. Male transgenic mice displayed no differences in anxiety, but their locomotor activity increased compared to non-transgenic littermates. Conclusion Our results revealed anxiety-related traits and locomotor differences between transgenic mice expressing constitutive active MAPKAPK5 and control littermates.

Characterization of a sensitive mouse Aβ40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

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Lu, Yanmei; Hoyte, Kwame; Montgomery, William H; Luk, Wilman; He, Dongping; Meilandt, William J; Zuchero, Y Joy Yu; Atwal, Jasvinder K; Scearce-Levie, Kimberly; Watts, Ryan J; DeForge, Laura E

2016-05-01

Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aβ40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aβ reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Wild-type and mutated IDH1/2 enzymes and therapy responses.

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Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

2018-04-01

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice

DEFF Research Database (Denmark)

Bang, Christian A; Bro, Susanne; Bartels, Emil D

2007-01-01

Wild-type mice normally do not develop atherosclerosis, unless fed cholic acid. Uremia is proinflammatory and increases atherosclerosis 6- to 10-fold in apolipoprotein E-deficient mice. This study examined the effect of uremia on lipoproteins, vascular inflammation, and atherosclerosis in wild...... in cholic acid-fed sham mice. The results suggest that moderate uremia neither induces aortic inflammation nor atherosclerosis in C57BL/6J mice despite increased LDL/HDL cholesterol ratio and altered HDL composition....

Metallothionein-I overexpression alters brain inflammation and stimulates brain repair in transgenic mice with astrocyte-targeted interleukin-6 expression

DEFF Research Database (Denmark)

Penkowa, Milena; Camats, Jordi; Giralt, Mercedes

2003-01-01

injury, such as a cryolesion, demonstrate a neuroprotective role of IL-6. Thus, the GFAP-IL-6 mice showed faster tissue repair and decreased oxidative stress and apoptosis compared with control litter-mate mice. The neuroprotective factors metallothionein-I+II (MT-I+II) were upregulated by the cryolesion...... the inflammatory response, decreased oxidative stress and apoptosis significantly, and increased brain tissue repair in comparison with either GFAP-IL-6 or control litter-mate mice. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors....

A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

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Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

2005-01-01

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

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François Huaux

Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

Diesel engine exhaust accelerates plaque formation in a mouse model of Alzheimer's disease.

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Hullmann, Maja; Albrecht, Catrin; van Berlo, Damiën; Gerlofs-Nijland, Miriam E; Wahle, Tina; Boots, Agnes W; Krutmann, Jean; Cassee, Flemming R; Bayer, Thomas A; Schins, Roel P F

2017-08-30

Increasing evidence from toxicological and epidemiological studies indicates that the central nervous system is an important target for ambient air pollutants. We have investigated whether long-term inhalation exposure to diesel engine exhaust (DEE), a dominant contributor to particulate air pollution in urban environments, can aggravate Alzheimer's Disease (AD)-like effects in female 5X Familial AD (5XFAD) mice and their wild-type female littermates. Following 3 and 13 weeks exposures to diluted DEE (0.95 mg/m 3 , 6 h/day, 5 days/week) or clean air (controls) behaviour tests were performed and amyloid-β (Aβ) plaque formation, pulmonary histopathology and systemic inflammation were evaluated. In a string suspension task, assessing for grip strength and motor coordination, 13 weeks exposed 5XFAD mice performed significantly less than the 5XFAD controls. Spatial working memory deficits, assessed by Y-maze and X-maze tasks, were not observed in association with the DEE exposures. Brains of the 3 weeks DEE-exposed 5XFAD mice showed significantly higher cortical Aβ plaque load and higher whole brain homogenate Aβ42 levels than the clean air-exposed 5XFAD littermate controls. After the 13 weeks exposures, with increasing age and progression of the AD-phenotype of the 5XFAD mice, DEE-related differences in amyloid pathology were no longer present. Immunohistochemical evaluation of lungs of the mice revealed no obvious genetic background-related differences in tissue structure, and the DEE exposure did not cause histopathological changes in the mice of both backgrounds. Luminex analysis of plasma cytokines demonstrated absence of sustained systemic inflammation upon DEE exposure. Inhalation exposure to DEE causes accelerated plaque formation and motor function impairment in 5XFAD transgenic mice. Our study provides further support that the brain is a relevant target for the effects of inhaled DEE and suggests that long-term exposure to this ubiquitous air

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

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Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

2010-01-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

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Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-03-01

1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanoloctanoloctanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.

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Li, Yu; Wong, Kimberly; Giles, Amber; Jiang, Jianwei; Lee, Jong Woo; Adams, Andrew C; Kharitonenkov, Alexei; Yang, Qin; Gao, Bin; Guarente, Leonard; Zang, Mengwei

2014-02-01

The hepatocyte-derived hormone fibroblast growth factor 21 (FGF21) is a hormone-like regulator of metabolism. The nicotinamide adenine dinucleotide-dependent deacetylase SIRT1 regulates fatty acid metabolism through multiple nutrient sensors. Hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese mice. We investigated mechanisms by which SIRT1 controls hepatic steatosis in mice. Liver-specific SIRT1 knockout (SIRT1 LKO) mice and their wild-type littermates (controls) were divided into groups that were placed on a normal chow diet, fasted for 24 hours, or fasted for 24 hours and then fed for 6 hours. Liver tissues were collected and analyzed by histologic examination, gene expression profiling, and real-time polymerase chain reaction assays. Human HepG2 cells were incubated with pharmacologic activators of SIRT1 (resveratrol or SRT1720) and mitochondrion oxidation consumption rate and immunoblot analyses were performed. FGF21 was overexpressed in SIRT1 LKO mice using an adenoviral vector. Energy expenditure was assessed by indirect calorimetry. Prolonged fasting induced lipid deposition in livers of control mice, but severe hepatic steatosis in SIRT1 LKO mice. Gene expression analysis showed that fasting up-regulated FGF21 in livers of control mice but not in SIRT1 LKO mice. Decreased hepatic and circulating levels of FGF21 in fasted SIRT1 LKO mice were associated with reduced hepatic expression of genes involved in fatty acid oxidation and ketogenesis, and increased expression of genes that control lipogenesis, compared with fasted control mice. Resveratrol or SRT1720 each increased the transcriptional activity of the FGF21 promoter (-2070/+117) and levels of FGF21 messenger RNA and protein in HepG2 cells. Surprisingly, SIRT1 LKO mice developed late-onset obesity with impaired whole-body energy expenditure. Hepatic overexpression of FGF21 in SIRT1 LKO mice increased the expression of genes that regulate fatty acid oxidation, decreased

Dietary Calcium and Dairy Modulation of Oxidative Stress and Mortality in aP2-Agouti and Wild-type Mice

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Antje Bruckbauer

2009-08-01

Full Text Available Oxidative and inflammatory stress have been implicated as major contributors to the aging process. Dietary Ca reduced both factors in short-term interventions, while milk exerted a greater effect than supplemental Ca. In this work, we examined the effects of life-long supplemental and dairy calcium on lifespan and life-span related biomarkers in aP2-agouti transgenic (model of diet-induced obesity and wild-type mice fed obesigenic diets until their death. These data demonstrate that dairy Ca exerts sustained effects resulting in attenuated adiposity, protection against age-related muscle loss and reduction of oxidative and inflammatory stress in both mouse strains. Although these effects did not alter maximum lifespan, they did suppress early mortality in wild-type mice, but not in aP2-agouti transgenic mice.

Effect of deuterium on the circadian period and metabolism in wild-type and tau mutant Syrian hamsters

NARCIS (Netherlands)

Oklejewicz, M; Hut, RA; Daan, S

2000-01-01

Homozygous tau mutant Syrian hamsters (tau-/-) have a free-running circadian period (tau) around 20 h and a proportionally higher metabolic rate compared with wild-type hamsters (tau+/+) with a period of circa 24 h. In this study, we applied deuterium oxide (D2O) to hamsters to test whether

Hypocretin/orexin loss changes the hypothalamic immune response.

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Tanaka, Susumu; Takizawa, Nae; Honda, Yoshiko; Koike, Taro; Oe, Souichi; Toyoda, Hiromi; Kodama, Tohru; Yamada, Hisao

2016-10-01

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy. Copyright © 2016 Elsevier Inc. All

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFδ/δ mouse model of amyotrophic lateral sclerosis

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Lambrechts Diether

2010-03-01

Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS. Investigating the molecular pathways to neurodegeneration in the VEGFδ/δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFδ/δ mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFδ/δ mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFδ/δ mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFδ/δ mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of

Normal social seeking behavior, hypoactivity and reduced exploratory range in a mouse model of Angelman syndrome

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Reiter Lawrence T

2011-01-01

Full Text Available Abstract Background Angelman syndrome (AS is a neurogenetic disorder characterized by severe developmental delay with mental retardation, a generally happy disposition, ataxia and characteristic behaviors such as inappropriate laughter, social-seeking behavior and hyperactivity. The majority of AS cases are due to loss of the maternal copy of the UBE3A gene. Maternal Ube3a deficiency (Ube3am-/p+, as well as complete loss of Ube3a expression (Ube3am-/p-, have been reproduced in the mouse model used here. Results Here we asked if two characteristic AS phenotypes - social-seeking behavior and hyperactivity - are reproduced in the Ube3a deficient mouse model of AS. We quantified social-seeking behavior as time spent in close proximity to a stranger mouse and activity as total time spent moving during exploration, movement speed and total length of the exploratory path. Mice of all three genotypes (Ube3am+/p+, Ube3am-/p+, Ube3am-/p- were tested and found to spend the same amount of time in close proximity to the stranger, indicating that Ube3a deficiency in mice does not result in increased social seeking behavior or social dis-inhibition. Also, Ube3a deficient mice were hypoactive compared to their wild-type littermates as shown by significantly lower levels of activity, slower movement velocities, shorter exploratory paths and a reduced exploratory range. Conclusions Although hyperactivity and social-seeking behavior are characteristic phenotypes of Angelman Syndrome in humans, the Ube3a deficient mouse model does not reproduce these phenotypes in comparison to their wild-type littermates. These phenotypic differences may be explained by differences in the size of the genetic defect as ~70% of AS patients have a deletion that includes several other genes surrounding the UBE3A locus.

Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

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Zhang, Min; Mongue-Din, Heloise; Martin, Daniel; Catibog, Norman; Smyrnias, Ioannis; Zhang, Xiaohong; Yu, Bin; Wang, Minshu; Brandes, Ralf P; Schröder, Katrin; Shah, Ajay M

2018-03-01

NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. © The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.

The effect of omega-3 fatty acids on central nervous system remyelination in fat-1 mice.

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Siegert, Elise; Paul, Friedemann; Rothe, Michael; Weylandt, Karsten H

2017-01-24

There is a large body of experimental evidence suggesting that omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are capable of modulating immune function. Some studies have shown that these PUFAs might have a beneficial effect in patients suffering form multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS). This could be due to increased n-3 PUFA-derived anti-inflammatory lipid mediators. In the present study we tested the effect of an endogenously increased n-3 PUFA status on cuprizone-induced CNS demyelination and remyelination in fat-1 mice versus their wild-type (wt) littermates. Fat-1 mice express an n-3 desaturase, which allows them to convert n-6 PUFAs into n-3 PUFAs. CNS lipid profiles in fat-1 mice showed a significant increase of eicosapentaenoic acid (EPA) levels but similar docosahexaenoic acid levels compared to wt littermates. This was also reflected in significantly higher levels of monohydroxy EPA metabolites such as 18-hydroxyeicosapentaenoic acid (18-HEPE) in fat-1 brain tissue. Feeding fat-1 mice and wt littermates 0.2% cuprizone for 5 weeks caused a similar degree of CNS demyelination in both groups; remyelination was increased in the fat-1 group after a recovery period of 2 weeks. However, at p = 0.07 this difference missed statistical significance. These results indicate that n-3 PUFAs might have a role in promotion of remyelination after toxic injury to CNS oligodendrocytes. This might occur either via modulation of the immune system or via a direct effect on oligodendrocytes or neurons through EPA-derived lipid metabolites such as 18-HEPE.

Lymphotropism and host responses during acute wild-type canine distemper virus infections in a highly susceptible natural host

DEFF Research Database (Denmark)

Nielsen, Line; Søgaard, Mette; Jensen, Trine Hammer

2009-01-01

The mechanisms behind the in vivo virulence of immunosuppressive wild-type Morbillivirus infections are still not fully understood. To investigate lymphotropism and host responses we have selected the natural host model of canine distemper virus (CDV) infection in mink. This model displays...

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

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Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Communication impairments in mice lacking Shank1: reduced levels of ultrasonic vocalizations and scent marking behavior.

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Markus Wöhr

Full Text Available Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1(-/- null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1(-/- mice as compared to wildtype Shank1(+/+ littermate controls. Shank1(-/- pups emitted fewer vocalizations than Shank1(+/+ pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1(-/- males deposited fewer scent marks in proximity to female urine than Shank1(+/+ males. Call emission in response to female urinary pheromones also differed between genotypes. Shank1(+/+ mice changed their calling pattern dependent on previous female interactions, while Shank1(-/- mice were unaffected, indicating a failure of Shank1(-/- males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1(-/- mice are consistent with a phenotype relevant to social communication deficits in autism.

Conditioned taste aversion memory and c-Fos induction are disrupted in RIIbeta-protein kinase A mutant mice.

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Koh, Ming Teng; Clarke, Sharon N D A; Spray, Kristina J; Thiele, Todd E; Bernstein, Ilene L

2003-07-14

The cAMP-dependent protein kinase (PKA) signaling pathway has been implicated in many forms of learning. The present studies examined conditioned taste aversion (CTA) learning, an amygdala-dependent task, in mice with a targeted disruption of a gene for a specific regulatory subunit of PKA (RIIbeta), which is selectively expressed in amygdala. Null mutant (RIIbeta(-/-)) mice and littermate controls (RIIbeta(+/+)) were tested for protein synthesis-independent short-term memory (STM) and protein synthesis-dependent long-term memory (LTM) for CTAs. The ability of the unconditioned stimulus (US) drug, LiCl, to induce c-Fos in regions thought to be important in this learning was also determined. RIIbeta(-/-) mice showed significant impairment in CTA memory when tested 24h after training (LTM). In contrast, STM was normal. With regard to the c-Fos response to LiCl, the US drug, significant elevations were evident in brainstem (nucleus of the solitary tract) and pontine (parabrachial nucleus) regions, in mutants as well as wild-type controls. However, in amygdala, elevations were seen in controls but were absent in the mutants. These findings suggest that disruption of PKA signaling interferes with LTM consolidation of CTA and that a possible mediator of this effect is interference with c-Fos expression in amygdala which may be necessary for CTA memory.

Economic Analysis of First-Line Treatment with Cetuximab or Panitumumab for RAS Wild-Type Metastatic Colorectal Cancer in England.

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Tikhonova, Irina A; Huxley, Nicola; Snowsill, Tristan; Crathorne, Louise; Varley-Campbell, Jo; Napier, Mark; Hoyle, Martin

2018-03-01

Combination therapies with cetuximab (Erbitux ® ; Merck Serono UK Ltd) and panitumumab (Vectibix ® ; Amgen UK Ltd) are shown to be less effective in adults with metastatic colorectal cancer who have mutations in exons 2, 3 and 4 of KRAS and NRAS oncogenes from the rat sarcoma (RAS) family. The objective of the study was to estimate the cost effectiveness of these drugs in patients with previously untreated RAS wild-type (i.e. non-mutated) metastatic colorectal cancer, not eligible for liver resection at baseline, from the UK National Health Service and Personal Social Services perspective. We constructed a partitioned survival model to evaluate the long-term costs and benefits of cetuximab and panitumumab combined with either FOLFOX (folinic acid, fluorouracil and oxaliplatin) or FOLFIRI (folinic acid, fluorouracil and irinotecan) vs. FOLFOX or FOLFIRI alone. The economic analysis was based on three randomised controlled trials. Costs and quality-adjusted life-years were discounted at 3.5% per annum. Based on the evidence available, both drugs fulfil the National Institute for Health and Care Excellence's end-of-life criteria. In the analysis, assuming discount prices for the drugs from patient access schemes agreed by the drug manufacturers with the Department of Health, predicted mean incremental cost-effectiveness ratios for cetuximab + FOLFOX, panitumumab + FOLFOX and cetuximab + FOLFIRI compared with chemotherapy alone appeared cost-effective at the National Institute for Health and Care Excellence's threshold of £50,000 per quality-adjusted life-year gained, applicable to end-of-life treatments. Cetuximab and panitumumab were recommended by the National Institute for Health and Care Excellence for patients with previously untreated RAS wild-type metastatic colorectal cancer, not eligible for liver resection at baseline, for use within the National Health Service in England. Both treatments are available via the UK Cancer Drugs Fund.

Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells

International Nuclear Information System (INIS)

Ito, Atsushi; Nakano, Hisako; Shinohara, Kunio

2010-01-01

The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells. (author)

Role of Peroxiredoxin III in the Pathogenesis of Pre-eclampsia as Evidenced in Mice

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Lianqin Li

2010-01-01

Full Text Available As a member of peroxiredoxin (Prx family, PrxIII has been demonstrated to play an important role in scavenging intracellular reactive oxygen species (ROS. Since PrxIII knockout mice exhibited oxidative stress in placentas resembling pathophysiologic changes in placentas of human pre-eclampsia, we measured blood pressure through the carotid artery and detected oxidative status by western blotting in pregnant mice. We did not notice hypertension in pregnant PrxIII knockout mice as compared with wild-type littermates, although endothelin-1 was overexpressed in PrxIII-deficient placentas. Our results indicate that PrxIII is not involved in pre-eclamptic development. Instead, PrxIII is an indispensable antioxidant in placentas where oxidative stress exists.

Age-related changes in body composition of bovine growth hormone transgenic mice.

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Palmer, Amanda J; Chung, Min-Yu; List, Edward O; Walker, Jennifer; Okada, Shigeru; Kopchick, John J; Berryman, Darlene E

2009-03-01

GH has a significant impact on body composition due to distinct anabolic and catabolic effects on lean and fat mass, respectively. Several studies have assessed body composition in mice expressing a GH transgene. Whereas all studies report enhanced growth of transgenic mice as compared with littermate controls, there are inconsistencies in terms of the relative proportion of lean mass to fat mass in these animals. The purpose of this study was to characterize the accumulation of adipose and lean mass with age and according to gender in a bovine (b) GH transgenic mouse line. Weight and body composition measurements were assessed in male and female bGH mice with corresponding littermate controls in the C57BL/6J genetic background. Body composition measurements began at 6 wk and continued through 1 yr of age. At the conclusion of the study, tissue weights were determined and triglyceride content was quantified in liver and kidney. Although body weights for bGH mice were significantly greater than their corresponding littermate controls at all time points, body composition measurements revealed an unexpected transition midway through analyses. That is, younger bGH mice had relatively more fat mass than nontransgenic littermates, whereas bGH mice became significantly leaner than controls by 4 months in males and 6 months in females. These results reveal the importance in timing and gender when conducting studies related to body composition or lean and fat tissue in GH transgenic mice or in other genetically manipulated mouse strains in which body composition may be impacted.

Slick (Kcnt2 Sodium-Activated Potassium Channels Limit Peptidergic Nociceptor Excitability and Hyperalgesia

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Danielle L Tomasello

2017-09-01

Full Text Available The Slick (Kcnt2 sodium-activated potassium (K Na channel is a rapidly gating and weakly voltage-dependent and sodium-dependent potassium channel with no clearly defined physiological function. Within the dorsal root ganglia (DRGs, we show Slick channels are exclusively expressed in small-sized and medium-sized calcitonin gene–related peptide (CGRP-containing DRG neurons, and a pool of channels are localized to large dense-core vesicles (LDCV-containing CGRP. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP-positive LDCV translocated to the neuronal membrane. Behavioral studies in Slick knockout (KO mice indicated increased basal heat detection and exacerbated thermal hyperalgesia compared with wild-type littermate controls during neuropathic and chronic inflammatory pain. Electrophysiologic recordings of DRG neurons from Slick KO mice revealed that Slick channels contribute to outward current, propensity to fire action potentials (APs, and to AP properties. Our data suggest that Slick channels restrain the excitability of CGRP-containing neurons, diminishing pain behavior after inflammation and injury.

PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle.

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Glenn C Rowe

Full Text Available Exercise confers numerous health benefits, many of which are thought to stem from exercise-induced mitochondrial biogenesis (EIMB in skeletal muscle. The transcriptional coactivator PGC-1α, a potent regulator of metabolism in numerous tissues, is widely believed to be required for EIMB. We show here that this is not the case. Mice engineered to lack PGC-1α specifically in skeletal muscle (Myo-PGC-1αKO mice retained intact EIMB. The exercise capacity of these mice was comparable to littermate controls. Induction of metabolic genes after 2 weeks of in-cage voluntary wheel running was intact. Electron microscopy revealed no gross abnormalities in mitochondria, and the mitochondrial biogenic response to endurance exercise was as robust in Myo-PGC-1αKO mice as in wildtype mice. The induction of enzymatic activity of the electron transport chain by exercise was likewise unperturbed in Myo-PGC-1αKO mice. These data demonstrate that PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle, in sharp contrast to the prevalent assumption in the field.

The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility.

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Escalante, Nichole K; Lemire, Paul; Cruz Tleugabulova, Mayra; Prescott, David; Mortha, Arthur; Streutker, Catherine J; Girardin, Stephen E; Philpott, Dana J; Mallevaey, Thierry

2016-12-12

The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1 -/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research. © 2016 Escalante et al.

Neonatal maternal deprivation response and developmental changes in gene expression revealed by hypothalamic gene expression profiling in mice.

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Feng Ding

Full Text Available Neonatal feeding problems are observed in several genetic diseases including Prader-Willi syndrome (PWS. Later in life, individuals with PWS develop hyperphagia and obesity due to lack of appetite control. We hypothesized that failure to thrive in infancy and later-onset hyperphagia are related and could be due to a defect in the hypothalamus. In this study, we performed gene expression microarray analysis of the hypothalamic response to maternal deprivation in neonatal wild-type and Snord116del mice, a mouse model for PWS in which a cluster of imprinted C/D box snoRNAs is deleted. The neonatal starvation response in both strains was dramatically different from that reported in adult rodents. Genes that are affected by adult starvation showed no expression change in the hypothalamus of 5 day-old pups after 6 hours of maternal deprivation. Unlike in adult rodents, expression levels of Nanos2 and Pdk4 were increased, and those of Pgpep1, Ndp, Brms1l, Mett10d, and Snx1 were decreased after neonatal deprivation. In addition, we compared hypothalamic gene expression profiles at postnatal days 5 and 13 and observed significant developmental changes. Notably, the gene expression profiles of Snord116del deletion mice and wild-type littermates were very similar at all time points and conditions, arguing against a role of Snord116 in feeding regulation in the neonatal period.

Altered social cognition in male BDNF heterozygous mice and following chronic methamphetamine exposure.

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Manning, Elizabeth E; van den Buuse, Maarten

2016-05-15

Growing clinical evidence suggests that persistent psychosis which occurs in methamphetamine users is closely related to schizophrenia. However, preclinical studies in animal models have focussed on psychosis-related behaviours following methamphetamine, and less work has been done to assess endophenotypes relevant to other deficits observed in schizophrenia. Altered social behaviour is a feature of both the negative symptoms and cognitive deficits in schizophrenia, and significantly impacts patient functioning. We recently found that brain-derived neurotrophic factor (BDNF) heterozygous mice show disrupted sensitization to methamphetamine, supporting other work suggesting an important role of this neurotrophin in the pathophysiology of psychosis and the neuronal response to stimulant drugs. In the current study, we assessed social and cognitive behaviours in methamphetamine-treated BDNF heterozygous mice and wildtype littermate controls. Following chronic methamphetamine exposure male wildtype mice showed a 50% reduction in social novelty preference. Vehicle-treated male BDNF heterozygous mice showed a similar impairment in social novelty preference, with a trend for no further disruption by methamphetamine exposure. Female mice were unaffected in this task, and no groups showed any changes in sociability or short-term spatial memory. These findings suggest that chronic methamphetamine alters behaviour relevant to disruption of social cognition in schizophrenia, supporting other studies which demonstrate a close resemblance between persistent methamphetamine psychosis and schizophrenia. Together these findings suggest that dynamic regulation of BDNF signalling is necessary to mediate the effects of methamphetamine on behaviours relevant to schizophrenia. Copyright © 2016 Elsevier B.V. All rights reserved.

Reduced alcohol consumption in mice lacking preprodynorphin.

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Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

2006-10-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Torpor patterns, arousal rates, and temporal organization of torpor entry in wildtype and UCP1-ablated mice.

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Oelkrug, R; Heldmaier, G; Meyer, C W

2011-01-01

In eutherian mammals, uncoupling protein 1 (UCP1) mediated non-shivering thermogenesis from brown adipose tissue (BAT) provides a mechanism through which arousal from torpor and hibernation is facilitated. In order to directly assess the magnitude by which the presence or absence of UCP1 affects torpor patterns, rewarming and arousal rates within one species we compared fasting induced torpor in wildtype (UCP1(+/+)) and UCP1-ablated mice (UCP(-/-)). Torpor was induced by depriving mice of food for up to 48 h and by a reduction of ambient temperature (T (a)) from 30 to 18°C at four different time points after 18, 24, 30 and 36 h of food deprivation. In most cases, torpor bouts occurred within 20 min after the switch in ambient temperature (30-18°C). Torpor bouts expressed during the light phase lasted 3-6 h while significantly longer bouts (up to 16 h) were observed when mice entered torpor during the dark phase. The degree of hypometabolism (5-22 ml h(-1)) and hypothermia (19.5-26.7°C) was comparable in wildtype and UCP1-ablated mice, and both genotypes were able to regain normothermia. In contrast to wildtype mice, UCP1-ablated mice did not display multiple torpor bouts per day and their peak rewarming rates from torpor were reduced by 50% (UCP1(+/+): 0.24 ± 0.08°C min(-1); UCP1(-/-): 0.12 ± 0.04°C min(-1)). UCP1-ablated mice therefore took significantly longer to rewarm from 25 to 32°C (39 vs. 70 min) and required 60% more energy for this process. Our results demonstrate the energetic benefit of functional BAT for rapid arousal from torpor. They also suggest that torpor entry and maintenance may be dependent on endogenous rhythms.

Photomorphogenic responses to UV radiation III: a comparative study of UVB effects on anthocyanin and flavonoid accumulation in wild-type and aurea mutant of tomato (Lycopersicon esculentum Mill.)

International Nuclear Information System (INIS)

Brandt, K.; Giannini, A.; Lercari, B.

1995-01-01

The UV-mediated induction of anthocyanin and UV-absorbing compounds was characterized in etiolated hypocotyls of wild-type and aurea (au) mutant tomato seedlings. Ultraviolet radiation induced significant increases of anthocyanin and UV-absorbing compounds in hypocotyls of the au mutant and of its isogenic wild-type, but the differences in the time courses of UV-induced pigment accumulation indicate that different photoregulatory mechanisms are involved for each of these two groups of pigments. It appears that prolonged presence of adequate levels of UVB (290-320 nm) energy and consequently the action of a specific UVB photoreceptor are indispensable for the photoinduction of anthocyanin accumulation in UV-irradiated hypocotyl of the au mutant that is missing the labile phytochrome pool. The large difference found between the wild-type and the au mutant strongly indicate the involvement of labile phytochrome as the primary functional photoreceptor for the photoinduction of anthocyanin accumulation in wild-type tomato hypocotyls. (author)

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

International Nuclear Information System (INIS)

Sedliakova, M.; Slezarikova, V.; Brozmanova, J.; Masek, F.; Bayerova, V.

1980-01-01

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7, proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival. We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP + ). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells given with potentially lethal doses of UV irradiation. (orig.)

Phase II marker-driven trial of panitumumab and chemotherapy in KRAS wild-type biliary tract cancer

DEFF Research Database (Denmark)

Jensen, L H; Lindebjerg, J; Ploen, J

2012-01-01

BACKGROUND: Combination chemotherapy has proven beneficial in biliary tract cancer and further improvements may be achieved by individualizing treatment based on biomarkers and by adding biological agents. We report the effect of chemotherapy with panitumumab as first-line therapy for KRAS wild....... Combination chemotherapy with panitumumab in patients with KRAS wild-type tumors met the efficacy criteria for future testing in a randomized trial....

Paternal spatial training enhances offspring's cognitive performance and synaptic plasticity in wild-type but not improve memory deficit in Alzheimer's mice.

Science.gov (United States)

Zhang, Shujuan; Li, Xiaoguang; Wang, Zhouyi; Liu, Yanchao; Gao, Yuan; Tan, Lu; Liu, Enjie; Zhou, Qiuzhi; Xu, Cheng; Wang, Xin; Liu, Gongping; Chen, Haote; Wang, Jian-Zhi

2017-05-08

Recent studies suggest that spatial training can maintain associative memory capacity in Tg2576 mice, but it is not known whether the beneficial effects can be inherited from the trained fathers to their offspring. Here, we exposed male wild-type and male 3XTg Alzheimer disease (AD) mice (3-m old) respectively to spatial training for one week and assessed the transgenerational effects in the F1 offspring when they were grown to 7-m old. We found that the paternal spatial training significantly enhanced progeny's spatial cognitive performance and synaptic transmission in wild-type mice. Among several synapse- or memory-associated proteins, we observed that the expression level of synaptotagmin 1 (SYT1) was significantly increased in the hippocampus of the paternally trained-offspring. Paternal training increased histone acetylation at the promoter of SYT1 in both fathers' and the offspring's hippocampus, and as well as in the fathers' sperm. Finally, paternal spatial training for one week did not improve memory and synaptic plasticity in 3XTg AD F1 offspring. Our findings suggest paternal spatial training for one week benefits the offspring's cognitive performance in wild-type mice with the mechanisms involving an enhanced transgenerational histone acetylation at SYT1 promoter.

Differential inhibition of ex-vivo tumor kinase activity by vemurafenib in BRAF(V600E and BRAF wild-type metastatic malignant melanoma.

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Andliena Tahiri

Full Text Available Treatment of metastatic malignant melanoma patients harboring BRAF(V600E has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

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Estes, Patricia S.; Boehringer, Ashley; Zwick, Rebecca; Tang, Jonathan E.; Grigsby, Brianna; Zarnescu, Daniela C.

2011-01-01

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders. PMID:21441568

Mapping the Conformation Space of Wildtype and Mutant H-Ras with a Memetic, Cellular, and Multiscale Evolutionary Algorithm.

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Rudy Clausen

2015-09-01

Full Text Available An important goal in molecular biology is to understand functional changes upon single-point mutations in proteins. Doing so through a detailed characterization of structure spaces and underlying energy landscapes is desirable but continues to challenge methods based on Molecular Dynamics. In this paper we propose a novel algorithm, SIfTER, which is based instead on stochastic optimization to circumvent the computational challenge of exploring the breadth of a protein's structure space. SIfTER is a data-driven evolutionary algorithm, leveraging experimentally-available structures of wildtype and variant sequences of a protein to define a reduced search space from where to efficiently draw samples corresponding to novel structures not directly observed in the wet laboratory. The main advantage of SIfTER is its ability to rapidly generate conformational ensembles, thus allowing mapping and juxtaposing landscapes of variant sequences and relating observed differences to functional changes. We apply SIfTER to variant sequences of the H-Ras catalytic domain, due to the prominent role of the Ras protein in signaling pathways that control cell proliferation, its well-studied conformational switching, and abundance of documented mutations in several human tumors. Many Ras mutations are oncogenic, but detailed energy landscapes have not been reported until now. Analysis of SIfTER-computed energy landscapes for the wildtype and two oncogenic variants, G12V and Q61L, suggests that these mutations cause constitutive activation through two different mechanisms. G12V directly affects binding specificity while leaving the energy landscape largely unchanged, whereas Q61L has pronounced, starker effects on the landscape. An implementation of SIfTER is made available at http://www.cs.gmu.edu/~ashehu/?q=OurTools. We believe SIfTER is useful to the community to answer the question of how sequence mutations affect the function of a protein, when there is an

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars

Science.gov (United States)

Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties. PMID:28191468

Screening and Expression of a Silicon Transporter Gene (Lsi1 in Wild-Type Indica Rice Cultivars

Directory of Open Access Journals (Sweden)

Mahbod Sahebi

2017-01-01

Full Text Available Silicon (Si is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars.

Science.gov (United States)

Sahebi, Mahbod; Hanafi, Mohamed M; Rafii, M Y; Azizi, Parisa; Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice

OpenAIRE

Walentiny, D. Matthew; Vann, Robert E.; Wiley, Jenny L.

2015-01-01

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ9 -tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with sim...

Aerobic Glycolysis in the Frontal Cortex Correlates with Memory Performance in Wild-Type Mice But Not the APP/PS1 Mouse Model of Cerebral Amyloidosis.

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Harris, Richard A; Tindale, Lauren; Lone, Asad; Singh, Olivia; Macauley, Shannon L; Stanley, Molly; Holtzman, David M; Bartha, Robert; Cumming, Robert C

2016-02-10

Aerobic glycolysis and lactate production in the brain plays a key role in memory, yet the role of this metabolism in the cognitive decline associated with Alzheimer's disease (AD) remains poorly understood. Here we examined the relationship between cerebral lactate levels and memory performance in an APP/PS1 mouse model of AD, which progressively accumulates amyloid-β. In vivo (1)H-magnetic resonance spectroscopy revealed an age-dependent decline in lactate levels within the frontal cortex of control mice, whereas lactate levels remained unaltered in APP/PS1 mice from 3 to 12 months of age. Analysis of hippocampal interstitial fluid by in vivo microdialysis revealed a significant elevation in lactate levels in APP/PS1 mice relative to control mice at 12 months of age. An age-dependent decline in the levels of key aerobic glycolysis enzymes and a concomitant increase in lactate transporter expression was detected in control mice. Increased expression of lactate-producing enzymes correlated with improved memory in control mice. Interestingly, in APP/PS1 mice the opposite effect was detected. In these mice, increased expression of lactate producing enzymes correlated with poorer memory performance. Immunofluorescent staining revealed localization of the aerobic glycolysis enzymes pyruvate dehydrogenase kinase and lactate dehydrogenase A within cortical and hippocampal neurons in control mice, as well as within astrocytes surrounding amyloid plaques in APP/PS1 mice. These observations collectively indicate that production of lactate, via aerobic glycolysis, is beneficial for memory function during normal aging. However, elevated lactate levels in APP/PS1 mice indicate perturbed lactate processing, a factor that may contribute to cognitive decline in AD. Lactate has recently emerged as a key metabolite necessary for memory consolidation. Lactate is the end product of aerobic glycolysis, a unique form of metabolism that occurs within certain regions of the brain. Here

Androgen receptor signals regulate UDP-glucuronosyltransferases in the urinary bladder: a potential mechanism of androgen-induced bladder carcinogenesis.

Science.gov (United States)

Izumi, Koji; Zheng, Yichun; Hsu, Jong-Wei; Chang, Chawnshang; Miyamoto, Hiroshi

2013-02-01

UDP-glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC-AR reduced mRNA expression of all the UGT1A subtypes (19-75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC-Vector. Of note were higher expression levels of UGT1As in SVHUC-Vector than in SVHUC-AR. In ARKO mice, all the Ugt1a subtypes were up-regulated, compared to wild-type littermates. In wild-type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild-type female mice had higher levels of Ugt1a than wild-type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen-mediated AR signals promote bladder carcinogenesis by down-regulating the expression of UGTs in the bladder. Copyright © 2011 Wiley Periodicals, Inc.

Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression Saccharomyces cerevisiae strains: The comparison of two quantitative methods

DEFF Research Database (Denmark)

Usaite, Renata; Wohlschlegel, James; Venable, John D.

2008-01-01

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild......-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches...... labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found...

Communication Impairments in Mice Lacking Shank1: Reduced Levels of Ultrasonic Vocalizations and Scent Marking Behavior

Science.gov (United States)

Wöhr, Markus; Roullet, Florence I.; Hung, Albert Y.; Sheng, Morgan; Crawley, Jacqueline N.

2011-01-01

Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1 −/− null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1 −/− mice as compared to wildtype Shank1 +/+ littermate controls. Shank1 −/− pups emitted fewer vocalizations than Shank1+/+ pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1 −/− males deposited fewer scent marks in proximity to female urine than Shank1+/+ males. Call emission in response to female urinary pheromones also differed between genotypes. Shank1+/+ mice changed their calling pattern dependent on previous female interactions, while Shank1 −/− mice were unaffected, indicating a failure of Shank1 −/− males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1 −/− mice are consistent with a phenotype relevant to social communication deficits in autism. PMID:21695253

Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

International Nuclear Information System (INIS)

Lopez, Carlos A.; Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

2007-01-01

Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G 1 arrest, increase in sub-G 1 fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios (± SE) of 1.5 (± 0.2) and 1.3 (± 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53

Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

Science.gov (United States)

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

2010-11-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

International Nuclear Information System (INIS)

Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S.; Damborsky, Jiri; Kuta Smatanova, Ivana

2011-01-01

Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution. Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2 1 2 1 2 1 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively

Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis

DEFF Research Database (Denmark)

Købmann, Brian Jensen; Solem, Christian; Jensen, Peter Ruhdal

2005-01-01

control on glycolysis and growth rate but high negative control on formate production. We find that PFK and PK have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK has high positive...... coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable to the control...... coefficient found for the entire operon; the strong positive control by PK almost cancels out the negative control by LDH on formate production. The analysis suggests that co-regulation of PFK and PK provides a very efficient way to regulate glycolysis, and co-regulating PK and LDH allows the cells...

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

Science.gov (United States)

Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

2015-01-01

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

Gene expression profiles of Bapx1 expressing FACS sorted cells from wildtype and Bapx1-EGFP null mouse embryos

Directory of Open Access Journals (Sweden)

Sumantra Chatterjee

2015-09-01

Full Text Available The data described in this article refers to Chatterjee et al. (2015 “In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column” (GEO GSE35649 [1]. Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb at a relevant developmental stage (E12.5, microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.

Preclinical efficacy of the MDM2 inhibitor RG7112 in MDM2 amplified and TP53 wild-type glioblastomas

Science.gov (United States)

Verreault, Maite; Schmitt, Charlotte; Goldwirt, Lauriane; Pelton, Kristine; Haidar, Samer; Levasseur, Camille; Guehennec, Jeremy; Knoff, David; Labussiere, Marianne; Marie, Yannick; Ligon, Azra H.; Mokhtari, Karima; Hoang-Xuan, Khe; Sanson, Marc; Alexander, Brian M; Wen, Patrick Y.; Delattre, Jean-Yves; Ligon, Keith L.; Idbaih, Ahmed

2016-01-01

Rationale p53 pathway alterations are key molecular events in glioblastoma (GBM). MDM2 inhibitors increase expression and stability of p53 and are presumed to be most efficacious in patients with TP53 wild-type and MDM2-amplified cancers. However, this biomarker hypothesis has not been tested in patients or patient-derived models for GBM. Methods We performed a preclinical evaluation of RG7112 MDM2 inhibitor, across a panel of 36 patient-derived GBM cell lines (PDCLs), each genetically characterized according to their P53 pathway status. We then performed a pharmacokinetic (PK) profiling of RG7112 distribution in mice and evaluated the therapeutic activity of RG7112 in orthotopic and subcutaneous GBM models. Results MDM2-amplified PDCLs were 44 times more sensitive than TP53 mutated lines that showed complete resistance at therapeutically attainable concentrations (avg. IC50 of 0.52 μM vs 21.9 μM). MDM4 amplified PDCLs were highly sensitive but showed intermediate response (avg. IC50 of 1.2 μM), whereas response was heterogeneous in TP53 wild-type PDCLs with normal MDM2/4 levels (avg. IC50 of 7.7 μM). In MDM2-amplified lines, RG7112 restored p53 activity inducing robust p21 expression and apoptosis. PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival. Conclusion These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2 amplified models suggests that additional markers of response to MDM2 inhibitors must be identified. PMID:26482041

Comparative Study on Growth Performance of Transgenic (Over-Expressed OsNHX1 and Wild-Type Nipponbare under Different Salinity Regimes

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Nurul Kahrani ISHAK

2015-11-01

Full Text Available Transgenic Nipponbare which over-expressed a Na+/H+ antiporter gene OsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

Cognitive impairments associated with alterations in synaptic proteins induced by the genetic loss of adenosine A2A receptors in mice.

Science.gov (United States)

Moscoso-Castro, Maria; López-Cano, Marc; Gracia-Rubio, Irene; Ciruela, Francisco; Valverde, Olga

2017-11-01

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A 2A adenosine receptor subtype (A 2A R) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A 2A R in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A 2A R constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A 2A R knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phase II Study of the Dual EGFR/HER3 Inhibitor Duligotuzumab (MEHD7945A) versus Cetuximab in Combination with FOLFIRI in Second-Line RAS Wild-Type Metastatic Colorectal Cancer.

Science.gov (United States)

Hill, Andrew G; Findlay, Michael P; Burge, Matthew E; Jackson, Christopher; Alfonso, Pilar Garcia; Samuel, Leslie; Ganju, Vinod; Karthaus, Meinolf; Amatu, Alessio; Jeffery, Mark; Bartolomeo, Maria Di; Bridgewater, John; Coveler, Andrew L; Hidalgo, Manuel; Kapp, Amy V; Sufan, Roxana I; McCall, Bruce B; Hanley, William D; Penuel, Elicia M; Pirzkall, Andrea; Tabernero, Josep

2018-05-15

Purpose: Duligotuzumab is a dual-action antibody directed against EGFR and HER3. Experimental Design: Metastatic colorectal cancer (mCRC) patients with KRAS ex2 wild-type received duligotuzumab or cetuximab and FOLFIRI until progression or intolerable toxicity. Mandatory tumor samples underwent mutation and biomarker analysis. Efficacy analysis was conducted in patients with RAS exon 2/3 wild-type tumors. Results: Of 134 randomly assigned patients, 98 had RAS ex2/3 wild-type. Duligotuzumab provided no progression-free survival (PFS) or overall survival (OS) benefit compared with cetuximab, although there was a trend for a lower objective response rate (ORR) in the duligotuzumab arm. No relationship was seen between PFS or ORR and ERBB3, NRG1, or AREG expression. There were fewer skin rash events for duligotuzumab but more diarrhea. Although the incidence of grade ≥3 AEs was similar, the frequency of serious AEs was higher for duligotuzumab. Conclusions: Duligotuzumab plus FOLFIRI did not appear to improve the outcomes in patients with RAS exon 2/3 wild-type mCRC compared with cetuximab + FOLFIRI. Clin Cancer Res; 24(10); 2276-84. ©2018 AACR . ©2018 American Association for Cancer Research.

Supplementation with Lactobacillus plantarum WCFS1 prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1-/Δ7 Mice

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Adriaan A Van Beek

2016-10-01

Full Text Available Although it is clear that probiotics improve intestinal barrier function, little is known about the effects of probiotics on the aging intestine. We investigated effects of 10-wk bacterial supplementation of Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, or Bifidobacterium breve DSM20213 on gut barrier and immunity in 16-week-old accelerated aging Ercc1-/Δ7 mice, which have a median lifespan of ~20wk, and their wild-type littermates. The colonic barrier in Ercc1-/Δ7 mice was characterized by a thin (<10µm mucus layer. L. plantarum prevented this decline in mucus integrity in Ercc1-/Δ7 mice, whereas B. breve exacerbated it. Bacterial supplementations affected the expression of immune-related genes, including Toll-like receptor 4. Regulatory T cell frequencies were increased in the mesenteric lymph nodes of L. plantarum- and L. casei-treated Ercc1-/Δ7 mice. L. plantarum- and L. casei-treated Ercc1-/Δ7 mice showed increased specific antibody production in a T cell-dependent immune response in vivo. By contrast, the effects of bacterial supplementation on wild-type control mice were negligible. Thus, supplementation with L. plantarum – but not with L. casei and B. breve – prevented the decline in the mucus barrier in Ercc1-/Δ7 mice. Our data indicate that age is an important factor influencing beneficial or detrimental effects of candidate probiotics. These findings also highlight the need for caution in translating beneficial effects of probiotics observed in young animals or humans to the elderly.

Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

Science.gov (United States)

Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

T-cell-dependent control of acute Giardia lamblia infections in mice.

Science.gov (United States)

Singer, S M; Nash, T E

2000-01-01

We have studied immune mechanisms responsible for control of acute Giardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice with G. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to control G. lamblia infections, as has been shown previously for G. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either alphabeta- or gammadelta-T-cell receptor (TCR)-expressing T cells, we show that the alphabeta-TCR-expressing T cells are required to control parasites but that the gammadelta-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection from G. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

Innate immune responses to obesity in cloned and wild-type domestic pig

DEFF Research Database (Denmark)

Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Stagsted, Jan

as a refined pig model for obesity-induced innate host responses by reducing pig-to-pig biological variation compared to wild-type (WT) pigs (n=19). Pigs were fed ad libitum with a high fat/high sucrose diet to induce obesity or kept lean on a restricted diet (60% of ad libitum intake) beginning at three...... months of age. mRNA expression levels were determined for 39 innate immune factors on a high-throughput qPCR system in samples from liver, abdominal fat, mesenteric fat and subcutaneous fat. Previous findings have suggested that cloning may affect certain phenotypic traits of pigs including basic...... concentrations and responsiveness of components of the innate immune system. Terminal body weights at 7½ - 9½ months of age were significantly higher for both (WT and cloned) obese groups compared to the lean groups. However, obese WT pigs weighed significantly more than obese cloned pigs (P

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

International Nuclear Information System (INIS)

Mitra, S.; Pal, B.C.; Foote, R.S.

1982-01-01

O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

Transcervical Inoculation with Chlamydia trachomatis Induces Infertility in HLA-DR4 Transgenic and Wild-Type Mice.

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Pal, Sukumar; Tifrea, Delia F; Zhong, Guangming; de la Maza, Luis M

2018-01-01

Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to control this epidemic with screening programs and antibiotic therapy have failed. Currently, a vaccine to prevent C. trachomatis infections is not available. In order to develop an animal model for evaluating vaccine antigens that can be applied to humans, we used C. trachomatis serovar D (strain UW-3/Cx) to induce infertility in mice whose major histocompatibility complex class II antigen was replaced with the human leukocyte antigen DR4 (HLA-DR4). Transcervical inoculation of medroxyprogesterone-treated HLA-DR4 transgenic mice with 5 × 10 5 C. trachomatis D inclusion forming units (IFU) induced a significant reduction in fertility, with a mean number of embryos/mouse of 4.4 ± 1.3 compared to 7.8 ± 0.5 for the uninfected control mice ( P < 0.05). A similar fertility reduction was elicited in the wild-type (WT) C57BL/6 mice (4.3 ± 1.4 embryos/mouse) compared to the levels of the WT controls (9.1 ± 0.4 embryos/mouse) ( P < 0.05). Following infection, WT mice mounted more robust humoral and cellular immune responses than HLA-DR4 mice. As determined by vaginal shedding, HLA-DR4 mice were more susceptible to a transcervical C. trachomatis D infection than WT mice. To assess if HLA-DR4 transgenic and WT mice could be protected by vaccination, 10 4 IFU of C. trachomatis D was delivered intranasally, and mice were challenged transcervically 6 weeks later with 5 × 10 5 IFU of C. trachomatis D. As determined by severity and length of vaginal shedding, WT C57BL/6 and HLA-DR4 mice were significantly protected by vaccination. The advantages and limitations of the HLA-DR4 transgenic mouse model for evaluating human C. trachomatis vaccine antigens are discussed. Copyright © 2017 American Society for Microbiology.

Oral carcinogenesis is not achieved in different carcinogen-treated PAI-1 transgenic and wild-type mouse models.

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Avgoustidis, Dimitris; Nisyrios, Themistoklis; Nkenke, Emeka; Lijnen, Roger; Ragos, Vassilis; Perrea, Despina; Donta, Ismini; Vaena, Apostolia; Yapijakis, Christos; Vairaktaris, Eleftherios

2012-01-01

In an effort to assess the role of plasminogen activator inhibitor-1 (PAI-1) in oral squamous cancer development and progression, two different carcinogen treatment protocols were conducted. Protocol I included mice from a PAI-1 transgenic (Tg) breed (n=56) and their wild-type (WT) counterparts (n=56), divided into one control group and two main experimental groups, treated with 7,12-dimethylbenz[a]anthracene (DMBA) for 8 and 16 weeks, respectively. Protocol II included the same number and types of animals and groups, which were similarly treated with 4-Nitroquinoline 1-oxide (4-NQO) in drinking water. Two drugs that affect plasma PAI-1 levels, enalapril and pravastatin, were administered to certain subgroups of animals in both protocols. None of the animals developed macroscopically-visible oral cancer lesions. Eleven animals under Protocol I and 52 animals under Protocol II died. Skin lesions were noted only in DMBA-treated animals (n=9). Almost all animals administered with 4-NQO developed alopecia and lost weight, while two of them developed stomach tumours, and one female mouse developed a large ovarian cyst. Transgenic mice may respond differently when used in well-established carcinogen models and oral carcinogenesis is hard to achieve in these rodents.

IFN-{gamma} enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease

DEFF Research Database (Denmark)

Baron, Rona; Nemirovsky, Anna; Harpaz, Idan

2008-01-01

the spatial learning and memory performance of the animals. In older mice, the effect of IFN-gamma is more pronounced in both wild-type mice and mice with Alzheimer's-like disease and is associated with neuroprotection. In addition, IFN-gamma reverses the increase in oligodendrogenesis observed in a mouse...... mechanisms can generate immunity to such deficits in neuronal repair. We demonstrate that in contrast to primarily innate immunity cytokines, such as interleukin-6 and tumor necrosis factor-alpha, the adaptive immunity cytokine IFN-gamma enhances neurogenesis in the dentate gyrus of adult mice and improves...

p53 functions as a cell cycle control protein in osteosarcomas.

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Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-11-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

p53 functions as a cell cycle control protein in osteosarcomas.

Science.gov (United States)

Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-01-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Images PMID:2233717

Lysogenic Streptococcus suis isolate SS2-4 containing prophage SMP showed increased mortality in zebra fish compared to the wild-type isolate.

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Fang Tang

Full Text Available Streptococcus suis (S. suis infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2 being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.

Gastric Medullary Carcinoma with Sporadic Mismatch Repair Deficiency and a TP53 R273C Mutation: An Unusual Case with Wild-Type BRAF

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Brett M. Lowenthal

2017-01-01

Full Text Available Medullary carcinoma has long been recognized as a subtype of colorectal cancer associated with microsatellite instability and Lynch syndrome. Gastric medullary carcinoma is a very rare neoplasm. We report a 67-year-old male who presented with a solitary gastric mass. Total gastrectomy revealed a well-demarcated, poorly differentiated carcinoma with an organoid growth pattern, pushing borders, and abundant peritumoral lymphocytic response. The prior cytology was cellular with immunohistochemical panel consistent with upper gastrointestinal/pancreaticobiliary origin. Overall, the histopathologic findings were consistent with gastric medullary carcinoma. A mismatch repair panel revealed a mismatch repair protein deficient tumor with loss of MLH1 and PMS2 expression. BRAF V600E immunostain (VE1 and BRAF molecular testing were negative, indicating a wild-type gene. Tumor sequencing of MLH1 demonstrated a wild-type gene, while our molecular panel identified TP53 c.817C>T (p.R273C mutation. These findings were compatible with a sporadic tumor. Given that morphologically identical medullary tumors often occur in Lynch syndrome, it is possible that mismatch repair loss is an early event in sporadic tumors with p53 mutation being a late event. Despite having wild-type BRAF, this tumor is sporadic and unrelated to Lynch syndrome. This case report demonstrates that coordinate ancillary studies are needed to resolve sporadic versus hereditary rare tumors.

A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

Science.gov (United States)

Cseke, Leland J; Talley, Sharon M

2012-02-20

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas(1-2). Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety(4) (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed. Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce

Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat.

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David F Razidlo

2010-07-01

Full Text Available Histone deacetylase (Hdac3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues

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Ahmed Al-Jazzar

2016-12-01

Full Text Available Mice harbouring a dentin matrix protein 1 (Dmp1 promoter-driven human diphtheria toxin (DT receptor (HDTR transgene (Tg have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2, was also found in many non-skeletal tissues in Tg mice; indicative of direct “off-target” DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.

Impaired spatial and contextual memory formation in galectin-1 deficient mice

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Sakaguchi Masanori

2011-09-01

Full Text Available Abstract Galectins are a 15 member family of carbohydrate-binding proteins that have been implicated in cancer, immunity, inflammation and development. While galectins are expressed in the central nervous system, little is known about their function in the adult brain. Previously we have shown that galectin-1 (gal-1 is expressed in the adult hippocampus, and, in particular, in putative neural stem cells in the subgranular zone. To evaluate how gal-1 might contribute to hippocampal memory function here we studied galectin-1 null mutant (gal-1-/- mice. Compared to their wildtype littermate controls, gal-1-/- mice exhibited impaired spatial learning in the water maze and contextual fear learning. Interestingly, tone fear conditioning was normal in gal-1-/- mice suggesting that loss of gal-1 might especially impact hippocampal learning and memory. Furthermore, gal-1-/- mice exhibited normal motor function, emotion and sensory processing in a battery of other behavioral tests, suggesting that non-mnemonic performance deficits are unlikely to account for the spatial and contextual learning deficits. Together, these data reveal a role for galectin-carbohydrate signalling in hippocampal memory function.

Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease.

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Gitte Krogh Nielsen

Full Text Available Niemann-Pick type C2 (NPC2 disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2(-/- mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2(-/- mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2(-/- and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2(-/- mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2(-/- animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the

Role of GSK-3β in the osteogenic differentiation of palatal mesenchyme.

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Emily R Nelson

Full Text Available INTRODUCTION: The function of Glycogen Synthase Kinases 3β (GSK-3β has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification. METHODS: Palates were harvested from GSK-3β, embryonic days 15.0-18.5 (e15.0-e18.5, and e15.5 Indian Hedgehog (Ihh null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists. RESULTS: GSK-3β null embryos displayed a 48 percent decrease (*p<0.05 in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1. CONCLUSIONS: Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways

A gene expression predictor of response to EGFR-targeted therapy stratifies progression-free survival to cetuximab in KRAS wild-type metastatic colorectal cancer

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Black Esther P

2009-05-01

Full Text Available Abstract Background The anti-EGFR monoclonal antibody cetuximab is used in metastatic colorectal cancer (CRC, and predicting responsive patients garners great interest, due to the high cost of therapy. Mutations in the KRAS gene occur in ~40% of CRC and are a negative predictor of response to cetuximab. However, many KRAS-wildtype patients do not benefit from cetuximab. We previously published a gene expression predictor of sensitivity to erlotinib, an EGFR inhibitor. The purpose of this study was to determine if this predictor could identify KRAS-wildtype CRC patients who will benefit from cetuximab therapy. Methods Microarray data from 80 metastatic CRC patients subsequently treated with cetuximab were extracted from the study by Khambata-Ford et al. The study included KRAS status, response, and PFS for each patient. The gene expression data were scaled and analyzed using our predictive model. An improved predictive model of response was identified by removing features in the 180-gene predictor that introduced noise. Results Forty-three of eighty patients were identified as harboring wildtype-KRAS. When the model was applied to these patients, the predicted-sensitive group had significantly longer PFS than the predicted-resistant group (median 88 days vs. 56 days; mean 117 days vs. 63 days, respectively, p = 0.008. Kaplan-Meier curves were also significantly improved in the predicted-sensitive group (p = 0.0059, HR = 0.4109. The model was simplified to 26 of the original 180 genes and this further improved stratification of PFS (median 147 days vs. 56.5 days in the predicted sensitive and resistant groups, respectively, p Conclusion Our model of sensitivity to EGFR inhibition stratified PFS following cetuximab in KRAS-wildtype CRC patients. This study represents the first true external validation of a molecular predictor of response to cetuximab in KRAS-WT metastatic CRC. Our model may hold clinical utility for identifying patients responsive

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels

OpenAIRE

Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl− channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl− channel activity of wild-type CFTR and delF508-CFTR mutant.The effects of n-alkanols like octanol on CFTR activity were measured by iodide (125I) efflux and patch-clamp techniques o...

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

Science.gov (United States)

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice

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Federico Brandalise

2017-01-01

Full Text Available Hericium erinaceus (Bull. Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice.

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Brandalise, Federico; Cesaroni, Valentina; Gregori, Andrej; Repetti, Margherita; Romano, Chiara; Orrù, Germano; Botta, Laura; Girometta, Carolina; Guglielminetti, Maria Lidia; Savino, Elena; Rossi, Paola

2017-01-01

Hericium erinaceus (Bull.) Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

Wild-type minimal inhibitory concentration distributions in bacteria of animal origin in Argentina

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Florencia L Pantozzi

Full Text Available The aim of this study was to determine the antimicrobial resistance profiles of indicator bacteria isolated from domestic animal feces. Minimal inhibitory concentration (MIC was determined by agar dilution. Interpretative criteria on the basis of wild-type MIC distributions and epidemiological cutoff values (ECOFF or ECV were used according to the 'European Committee on Antimicrobial Susceptibility Testing' (EUCAST data. Results from 237 isolates of Escherichia coli showed reduced susceptibility for ampicillin, streptomycin and tetracycline, the antimicrobials commonly used in intensive breeding of pigs and hens. Regarding all the species of the genus Enterococcus spp., there are only ECOFF or ECV for vancomycin. Of the 173 Enterococcus spp. isolated, only one showed reduced susceptibility to vancomycin and was classified as 'non-wild-type' (NWT population. This is the first report in Argentina showing data of epidemiological cutoff values in animal bacteria.

Dietary fish oil, and to a lesser extent the fat-1 transgene, increases astrocyte activation in response to intracerebroventricular amyloid-β 1-40 in mice.

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Hopperton, Kathryn E; James, Nicholas C E; Mohammad, Dana; Irfan, Maha; Bazinet, Richard P

2017-11-07

Increases in astrocytes and one of their markers, glial fibrillary acidic protein (GFAP) have been reported in the brains of patients with Alzheimer's disease (AD). N-3 polyunsaturated fatty acids (PUFA) modulate neuroinflammation in animal models; however, their effect on astrocytes is unclear. Fat-1 mice and their wildtype littermates were fed either a fish oil diet or a safflower oil diet deprived of n-3 PUFA. At 12 weeks, mice underwent intracerebroventricular infusion of amyloid-β 1-40. Astrocyte phenotype in the hippocampus was assessed at baseline and 10 days post-surgery using immunohistochemistry with various microscopy and image analysis techniques. GFAP increased in all groups in response to amyloid-β, with a greater increase in fish oil-fed mice than either fat-1 or wildtype safflower oil-fed mice. Astrocytes in this group were also more hypertrophic, suggesting increased activation. Both fat-1- and fish oil-fed mice had greater increases in branch number and length in response to amyloid-β infusion than wildtype safflower animals. Fish oil feeding, and to a lesser extent the fat-1 transgene, enhances the astrocyte activation phenotype in response to amyloid-β 1-40. Astrocytes in mice fed fish oil were more activated in response to amyloid-β than in fat-1 mice despite similar levels of hippocampal n-3 PUFA, which suggests that other fatty acids or dietary factors contribute to this effect.

Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

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Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

2002-01-01

It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

Primary tumor location predicts poor clinical outcome with cetuximab in RAS wild-type metastatic colorectal cancer.

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Kim, Dalyong; Kim, Sun Young; Lee, Ji Sung; Hong, Yong Sang; Kim, Jeong Eun; Kim, Kyu-Pyo; Kim, Jihun; Jang, Se Jin; Yoon, Young-Kwang; Kim, Tae Won

2017-11-23

In metastatic colorectal cancer, the location of the primary tumor has been suggested to have biological significance. In this study, we investigated whether primary tumor location affects cetuximab efficacy in patients with RAS wild-type metastatic colorectal cancer. Genotyping by the SequenomMassARRAY technology platform (OncoMap) targeting KRAS, NRAS, PIK3CA, and BRAF was performed in tumors from 307 patients who had been given cetuximab as salvage treatment. Tumors with mutated RAS (KRAS or NRAS; n = 127) and those with multiple primary location (n = 10) were excluded. Right colon cancer was defined as a tumor located in the proximal part to splenic flexure. A total of 170 patients were included in the study (right versus left, 23 and 147, respectively). Patients with right colon cancer showed more mutated BRAF (39.1% vs. 5.4%), mutated PIK3CA (13% vs. 1.4%), poorly differentiated tumor (17.4% vs. 3.4%), and peritoneal involvement (26.1% vs. 8.8%) than those with left colon and rectal cancer. Right colon cancer showed poorer progression-free survival (2.0 vs.5.0 months, P = 0.002) and overall survival (4.1 months and 13.0 months, P < 0.001) than the left colon and rectal cancer. By multivariable analysis, BRAF mutation, right colon primary, poorly differentiated histology, and peritoneal involvement were associated with risk of death. In RAS wild-type colon cancer treated with cetuximab as salvage treatment, right colon primary was associated with poorer survival outcomes than left colon and rectal cancer.

Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands.

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Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta

2011-02-01

Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

The use of lac-type promoters in control analysis

DEFF Research Database (Denmark)

Jensen, Peter Ruhdal; Westerhoff, H. v.; Michelsen, Ole

1993-01-01

experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times...... the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lac Y mutation facilitated...

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

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Marquet-de Rougé, Perrine; Clamagirand, Christine; Facchinetti, Patricia; Rose, Christiane; Sargueil, Françoise; Guihenneuc-Jouyaux, Chantal; Cynober, Luc; Moinard, Christophe; Allinquant, Bernadette

2013-10-01

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice

NARCIS (Netherlands)

Boelens, J. J.; van der Poll, T.; Zaat, S. A.; Murk, J. L.; Weening, J. J.; Dankert, J.

2000-01-01

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous

Electrotransformation and expression of cellulase genes in wild-type Lactobacillus reuteri.

Science.gov (United States)

Li, Wang; Yang, Ming-Ming; Zhang, Guang-Qin; He, Wan-Ling; Li, Yuan-Xiao; Chen, Yu-Lin

2012-01-01

Two cellulase genes, Cel15 and Cel73, were amplified from Bacillus subtilis genome DNA in a previous study. Two integrative vectors, pLEM4153 and pLEM4154, containing the genes Cel15 and Cel73, respectively, were constructed and successfully electroporated into the wild-type Lactobacillus reuteri which was isolated from chick guts through an optimized procedure. Two recombinant L. reuteri were selected from a Man, Rogosa, and Sharp (MRS) plate with 10 µg/ml erythromycin, and named L. reuteri XNY-Cel15 and L. reuteri XNY-Cel73, respectively. To verify the transcription and expression of the two cellulase genes in the recombinant L. reuteri strains, the mRNA relative quantity (RQ) and the cellulase activity were determined. The mRNA RQ of Cel15 in L. reuteri XNY-Cel15 is 1,8849.5, and that of Cel73 in L. reuteri XNY-Cel73 is 1,388, and the cellulase activity of the modified MRS broth cultured with L. reuteri XNY-Cel15 was 0.158 U/ml, whereas that with L. reuteri XNY-Cel73 was 0.15 U/ml. Copyright © 2012 S. Karger AG, Basel.

Long-term survival after a favorable response to anti-EGFR antibody plus chemotherapy to treat bone marrow metastasis: a case report of KRAS-wildtype rectal cancer

Directory of Open Access Journals (Sweden)

Nakamura S

2017-02-01

Full Text Available Sho Nakamura, Tadahisa Fukui, Shuhei Suzuki, Hiroyuki Takeda, Kaname Watanabe, Takashi Yoshioka Department of Clinical Oncology, Yamagata University Faculty of Medicine, Yamagata, Japan Abstract: Bone marrow metastasis is a rare consequence of colorectal cancer that results in a poor prognosis; few reports describe a favorable response to doublet chemotherapy combined with targeted therapy, which is currently the standard treatment. We experienced a case where anti-epidermal growth factor receptor (EGFR antibody produced a marked anti-tumor response to bone marrow metastasis that led to long-term survival. A 51-year-old man was diagnosed with a primary KRAS-wildtype rectal cancer with multiple metastases, including the bone marrow. Disease control was achieved for 10.8 months following chemotherapy with a modified FOLFOX6 regimen combined with an anti-EGFR antibody. He died of cancer 22.7 and 16.6 months after disease onset and first-line chemotherapy, respectively. This case shows that early tumor shrinkage and deepness of response to the anti-EGFR antibody were observed even in a patient with bone marrow metastasis. Anti-EGFR antibody therapy should therefore be considered even when a patient’s medical condition appears to be poor owing to bone marrow metastasis. Moreover, tumors that are likely to be sensitive to chemotherapy, such as RAS-wildtype colorectal cancers, can be considered for anti-EGFR antibody therapy even if the patient is considered unfit for chemotherapy. Keywords: colorectal cancer, anti-epidermal growth factor receptor antibody, molecular targeted therapies, disseminated intravascular coagulation, standard of care

Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

DEFF Research Database (Denmark)

Andersen, H.W.; Pedersen, M.B.; Hammer, Karin

2001-01-01

enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C- LDH(F1)J=-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate...

Effects of Specific Multi-Nutrient Enriched Diets on Cerebral Metabolism, Cognition and Neuropathology in AβPPswe-PS1dE9 Mice

Science.gov (United States)

Jansen, Diane; Zerbi, Valerio; Arnoldussen, Ilse A. C.; Wiesmann, Maximilian; Rijpma, Anne; Fang, Xiaotian T.; Dederen, Pieter J.; Mutsaers, Martina P. C.; Broersen, Laus M.; Lütjohann, Dieter; Miller, Malgorzata; Joosten, Leo A. B.; Heerschap, Arend; Kiliaan, Amanda J.

2013-01-01

Recent studies have focused on the use of multi-nutrient dietary interventions in search of alternatives for the treatment and prevention of Alzheimer's disease (AD). In this study we investigated to which extent long-term consumption of two specific multi-nutrient diets can modulate AD-related etiopathogenic mechanisms and behavior in 11-12-month-old AβPPswe-PS1dE9 mice. Starting from 2 months of age, male AβPP-PS1 mice and wild-type littermates were fed either a control diet, the DHA+EPA+UMP (DEU) diet enriched with uridine monophosphate (UMP) and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), or the Fortasyn® Connect (FC) diet enriched with the DEU diet plus phospholipids, choline, folic acid, vitamins and antioxidants. We performed behavioral testing, proton magnetic resonance spectroscopy, immunohistochemistry, biochemical analyses and quantitative real-time PCR to gain a better understanding of the potential mechanisms by which these multi-nutrient diets exert protective properties against AD. Our results show that both diets were equally effective in changing brain fatty acid and cholesterol profiles. However, the diets differentially affected AD-related pathologies and behavioral measures, suggesting that the effectiveness of specific nutrients may depend on the dietary context in which they are provided. The FC diet was more effective than the DEU diet in counteracting neurodegenerative aspects of AD and enhancing processes involved in neuronal maintenance and repair. Both diets elevated interleukin-1β mRNA levels in AβPP-PS1 and wild-type mice. The FC diet additionally restored neurogenesis in AβPP-PS1 mice, decreased hippocampal levels of unbound choline-containing compounds in wild-type and AβPP-PS1 animals, suggesting diminished membrane turnover, and decreased anxiety-related behavior in the open field behavior. In conclusion, the current data indicate that specific multi-nutrient diets can influence AD

Effects of specific multi-nutrient enriched diets on cerebral metabolism, cognition and neuropathology in AβPPswe-PS1dE9 mice.

Directory of Open Access Journals (Sweden)

Diane Jansen

Full Text Available Recent studies have focused on the use of multi-nutrient dietary interventions in search of alternatives for the treatment and prevention of Alzheimer's disease (AD. In this study we investigated to which extent long-term consumption of two specific multi-nutrient diets can modulate AD-related etiopathogenic mechanisms and behavior in 11-12-month-old AβPPswe-PS1dE9 mice. Starting from 2 months of age, male AβPP-PS1 mice and wild-type littermates were fed either a control diet, the DHA+EPA+UMP (DEU diet enriched with uridine monophosphate (UMP and the omega-3 fatty acids docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA, or the Fortasyn® Connect (FC diet enriched with the DEU diet plus phospholipids, choline, folic acid, vitamins and antioxidants. We performed behavioral testing, proton magnetic resonance spectroscopy, immunohistochemistry, biochemical analyses and quantitative real-time PCR to gain a better understanding of the potential mechanisms by which these multi-nutrient diets exert protective properties against AD. Our results show that both diets were equally effective in changing brain fatty acid and cholesterol profiles. However, the diets differentially affected AD-related pathologies and behavioral measures, suggesting that the effectiveness of specific nutrients may depend on the dietary context in which they are provided. The FC diet was more effective than the DEU diet in counteracting neurodegenerative aspects of AD and enhancing processes involved in neuronal maintenance and repair. Both diets elevated interleukin-1β mRNA levels in AβPP-PS1 and wild-type mice. The FC diet additionally restored neurogenesis in AβPP-PS1 mice, decreased hippocampal levels of unbound choline-containing compounds in wild-type and AβPP-PS1 animals, suggesting diminished membrane turnover, and decreased anxiety-related behavior in the open field behavior. In conclusion, the current data indicate that specific multi-nutrient diets can

Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

Science.gov (United States)

Suchland, Robert J; Jeffrey, Brendan M; Xia, Minsheng; Bhatia, Ajay; Chu, Hencelyn G; Rockey, Daniel D; Stamm, Walter E

2008-12-01

Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

Efficient mannitol production by wild-type Lactobacillus reuteri CRL 1101 is attained at constant pH using a simplified culture medium.

Science.gov (United States)

Ortiz, Maria Eugenia; Raya, Raúl R; Mozzi, Fernanda

2015-10-01

Mannitol is a natural polyol with multiple industrial applications. In this work, mannitol production by Lactobacillus reuteri CRL 1101 was studied at free- and controlled-pH (6.0-4.8) fermentations using a simplified culture medium containing yeast and beef extracts and sugarcane molasses. The activity of mannitol 2-dehydrogenase (MDH), the enzyme responsible for mannitol synthesis, was determined. The effect of the initial biomass concentration was further studied. Mannitol production (41.5 ± 1.1 g/l), volumetric productivity (Q Mtl 1.73 ± 0.05 g/l h), and yield (Y Mtl 105 ± 11 %) were maximum at pH 5.0 after 24 h while the highest MDH activity (1.66 ± 0.09 U/mg protein) was obtained at pH 6.0. No correlation between mannitol production and MDH activity was observed when varying the culture pH. The increase (up to 2000-fold) in the initial biomass concentration did not improve mannitol formation after 24 h although a 2-fold higher amount was produced at 8 h using 1 or 2 g cell dry weight/l comparing to the control (0.001 g cell dry weight/l). Finally, mannitol isolation under optimum fermentation conditions was achieved. The mannitol production obtained in this study is the highest reported so far by a wild-type L. reuteri strain and, more interestingly, using a simplified culture medium.

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

Science.gov (United States)

Guan, Wenna; Zhao, Hui; Lu, Xuefeng; Wang, Cong; Yang, Menglong; Bai, Fali

2011-11-11

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work. Copyright © 2011 Elsevier B.V. All rights reserved.

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) - Differences between vaccinated and wild-type virus exposed dogs.

Science.gov (United States)

Perrone, Danielle; Bender, Scott; Niewiesk, Stefan

2010-07-01

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals.

Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain.

Science.gov (United States)

Scherer, Thomas; Lindtner, Claudia; O'Hare, James; Hackl, Martina; Zielinski, Elizabeth; Freudenthaler, Angelika; Baumgartner-Parzer, Sabina; Tödter, Klaus; Heeren, Joerg; Krššák, Martin; Scheja, Ludger; Fürnsinn, Clemens; Buettner, Christoph

2016-06-01

Hepatic steatosis is common in obesity and insulin resistance and results from a net retention of lipids in the liver. A key mechanism to prevent steatosis is to increase secretion of triglycerides (TG) packaged as VLDLs. Insulin controls nutrient partitioning via signaling through its cognate receptor in peripheral target organs such as liver, muscle, and adipose tissue and via signaling in the central nervous system (CNS) to orchestrate organ cross talk. While hepatic insulin signaling is known to suppress VLDL production from the liver, it is unknown whether brain insulin signaling independently regulates hepatic VLDL secretion. Here, we show that in conscious, unrestrained male Sprague Dawley rats the infusion of insulin into the third ventricle acutely increased hepatic TG secretion. Chronic infusion of insulin into the CNS via osmotic minipumps reduced the hepatic lipid content as assessed by noninvasive (1)H-MRS and lipid profiling independent of changes in hepatic de novo lipogenesis and food intake. In mice that lack the insulin receptor in the brain, hepatic TG secretion was reduced compared with wild-type littermate controls. These studies identify brain insulin as an important permissive factor in hepatic VLDL secretion that protects against hepatic steatosis. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Compositional and Functional Differences in the Human Gut Microbiome Correlate with Clinical Outcome following Infection with Wild-Type Salmonella enterica Serovar Typhi.

Science.gov (United States)

Zhang, Yan; Brady, Arthur; Jones, Cheron; Song, Yang; Darton, Thomas C; Jones, Claire; Blohmke, Christoph J; Pollard, Andrew J; Magder, Laurence S; Fasano, Alessio; Sztein, Marcelo B; Fraser, Claire M

2018-05-08

Insights into disease susceptibility as well as the efficacy of vaccines against typhoid and other enteric pathogens may be informed by better understanding the relationship between the effector immune response and the gut microbiota. In the present study, we characterized the composition (16S rRNA gene profiling) and function (RNA sequencing [RNA-seq]) of the gut microbiota following immunization and subsequent exposure to wild-type Salmonella enterica serovar Typhi in a human challenge model to further investigate the central hypothesis that clinical outcomes may be linked to the gut microbiota. Metatranscriptome analysis of longitudinal stool samples collected from study subjects revealed two stable patterns of gene expression for the human gut microbiota, dominated by transcripts from either Methanobrevibacter or a diverse representation of genera in the Firmicutes phylum. Immunization with one of two live oral attenuated vaccines against S.  Typhi had minimal effects on the composition or function of the gut microbiota. It was observed that subjects harboring the methanogen-dominated transcriptome community at baseline displayed a lower risk of developing symptoms of typhoid following challenge with wild-type S.  Typhi. Furthermore, genes encoding antioxidant proteins, metal homeostasis and transport proteins, and heat shock proteins were expressed at a higher level at baseline or after challenge with S.  Typhi in subjects who did not develop symptoms of typhoid. These data suggest that functional differences relating to redox potential and ion homeostasis in the gut microbiota may impact clinical outcomes following exposure to wild-type S.  Typhi. IMPORTANCE S.  Typhi is a significant cause of systemic febrile morbidity in settings with poor sanitation and limited access to clean water. It has been demonstrated that the human gut microbiota can influence mucosal immune responses, but there is little information available on the impact of the human gut

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

Science.gov (United States)

Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

2009-01-01

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice.

Science.gov (United States)

Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena; Pizzorusso, Tommaso

2017-06-15

CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60-80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. © The Author 2017. Published by Oxford University Press.

Anhedonic behavior in cryptochrome 2-deficient mice is paralleled by altered diurnal patterns of amygdala gene expression.

Science.gov (United States)

Savalli, Giorgia; Diao, Weifei; Berger, Stefanie; Ronovsky, Marianne; Partonen, Timo; Pollak, Daniela D

2015-07-01

Mood disorders are frequently paralleled by disturbances in circadian rhythm-related physiological and behavioral states and genetic variants of clock genes have been associated with depression. Cryptochrome 2 (Cry2) is one of the core components of the molecular circadian machinery which has been linked to depression, both, in patients suffering from the disease and animal models of the disorder. Despite this circumstantial evidence, a direct causal relationship between Cry2 expression and depression has not been established. Here, a genetic mouse model of Cry2 deficiency (Cry2 (-/-) mice) was employed to test the direct relevance of Cry2 for depression-like behavior. Augmented anhedonic behavior in the sucrose preference test, without alterations in behavioral despair, was observed in Cry2 (-/-) mice. The novelty suppressed feeding paradigm revealed reduced hyponeophagia in Cry2 (-/-) mice compared to wild-type littermates. Given the importance of the amygdala in the regulation of emotion and their relevance for the pathophysiology of depression, potential alterations in diurnal patterns of basolateral amygdala gene expression in Cry2 (-/-) mice were investigated focusing on core clock genes and neurotrophic factor systems implicated in the pathophysiology of depression. Differential expression of the clock gene Bhlhe40 and the neurotrophic factor Vegfb were found in the beginning of the active (dark) phase in Cry2 (-/-) compared to wild-type animals. Furthermore, amygdala tissue of Cry2 (-/-) mice contained lower levels of Bdnf-III. Collectively, these results indicate that Cry2 exerts a critical role in the control of depression-related emotional states and modulates the chronobiological gene expression profile in the mouse amygdala.

Affective and cognitive behavior in the alpha-galactosidase A deficient mouse model of Fabry disease.

Directory of Open Access Journals (Sweden)

Lukas Hofmann

Full Text Available Fabry disease is an X-linked inherited lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3 due to α-galactosidase A (α-Gal A deficiency. Fabry patients frequently report of anxiety, depression, and impaired cognitive function. We characterized affective and cognitive phenotype of male mice with α-Gal A deficiency (Fabry KO and compared results with those of age-matched male wildtype (WT littermates. Young (3 months and old (≥ 18 months mice were tested in the naïve state and after i.pl. injection of complete Freund`s adjuvant (CFA as an inflammatory pain model. We used the elevated plus maze (EPM, the light-dark box (LDB and the open field test (OF to investigate anxiety-like behavior. The forced swim test (FST and Morris water maze (MWM were applied to assess depressive-like and learning behavior. The EPM test revealed no intergroup difference for anxiety-like behavior in naïve young and old Fabry KO mice compared to WT littermates, except for longer time spent in open arms of the EPM for young WT mice compared to young Fabry KO mice (p<0.05. After CFA injection, young Fabry KO mice showed increased anxiety-like behavior compared to young WT littermates (p<0.05 and naïve young Fabry KO mice (p<0.05 in the EPM as reflected by shorter time spent in EPM open arms. There were no relevant differences in the LDB and the OF test, except for longer time spent in the center zone of the OF by young WT mice compared to young Fabry KO mice (p<0.05. Complementary to this, depression-like and learning behavior were not different between genotypes and age-groups, except for the expectedly lower memory performance in older age-groups compared to young mice. Our results indicate that genetic influences on affective and cognitive symptoms in FD may be of subordinate relevance, drawing attention to potential influences of environmental and epigenetic factors.

Genotype-temperature interaction in the regulation of development, growth, and morphometrics in wild-type, and growth-hormone transgenic coho salmon.

Directory of Open Access Journals (Sweden)

Mare Lõhmus

2010-04-01

Full Text Available The neuroendocrine system is an important modulator of phenotype, directing cellular genetic responses to external cues such as temperature. Behavioural and physiological processes in poikilothermic organisms (e.g. most fishes, are particularly influenced by surrounding temperatures.By comparing the development and growth of two genotypes of coho salmon (wild-type and transgenic with greatly enhanced growth hormone production at six different temperatures, ranging between 8 degrees and 18 degrees C, we observed a genotype-temperature interaction and possible trend in directed neuroendocrine selection. Differences in growth patterns of the two genotypes were compared by using mathematical models, and morphometric analyses of juvenile salmon were performed to detect differences in body shape. The maximum hatching and alevin survival rates of both genotypes occurred at 12 degrees C. At lower temperatures, eggs containing embryos with enhanced GH production hatched after a shorter incubation period than wild-type eggs, but this difference was not apparent at and above 16 degrees C. GH transgenesis led to lower body weights at the time when the yolk sack was completely absorbed compared to the wild genotype. The growth of juvenile GH-enhanced salmon was to a greater extent stimulated by higher temperatures than the growth of the wild-type. Increased GH production significantly influenced the shape of the salmon growth curves.Growth hormone overexpression by transgenesis is able to stimulate the growth of coho salmon over a wide range of temperatures. Temperature was found to affect growth rate, survival, and body morphology between GH transgenic and wild genotype coho salmon, and differential responses to temperature observed between the genotypes suggests they would experience different selective forces should they ever enter natural ecosystems. Thus, GH transgenic fish would be expected to differentially respond and adapt to shifts in environmental

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

DEFF Research Database (Denmark)

Röpke, M; Hald, J; Guldberg, Per

1996-01-01

p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules...

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas

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Seongjoon Kang

2018-05-01

Full Text Available Chlamydomonas reinhardtii (Chlamydomonas strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa. Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations.

The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific.

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Stefanie L Morgan

Full Text Available The role for royal jelly (RJ in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species.

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus.

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Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter; Jensen, Trine Hammer; Jensen, Tove Dannemann; Aasted, Bent; Blixenkrone-Møller, Merete

2009-07-30

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV. The DNA vaccine-induced immunity protected the natural host against disease development.

Absorption and emission spectroscopic characterisation of combined wildtype LOV1-LOV2 domain of phot from Chlamydomonas reinhardtii.

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Song, S-H; Dick, B; Zirak, P; Penzkofer, A; Schiereis, T; Hegemann, P

2005-10-03

An absorption and emission spectroscopic characterisation of the combined wild-type LOV1-LOV2 domain string (abbreviated LOV1/2) of phot from the green alga Chlamydomonas reinhardtii is carried out at pH 8. A LOV1/2-MBP fusion protein (MBP=maltose binding protein) and LOV1/2 with a His-tag at the C-terminus (LOV1/2-His) expressed in an Escherichia coli strain are investigated. Blue-light photo-excitation generates a non-fluorescent intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm). The photo-cycle dynamics is studied by dark-state absorption and fluorescence measurement, by following the temporal absorption and emission changes under blue and violet light exposure, and by measuring the temporal absorption and fluorescence recovery after light exposure. The fluorescence quantum yield, phi(F), of the dark adapted samples is phi(F)(LOV1/2-His) approximately 0.15 and phi(F)(LOV1/2-MBP) approximately 0.17. A bi-exponential absorption recovery after light exposure with a fast (in the several 10-s range) and a slow component (in the near 10-min range) are resolved. The quantum yield of photo-adduct formation, phi(Ad), is extracted from excitation intensity dependent absorption measurements. It decreases somewhat with rising excitation intensity. The behaviour of the combined wildtype LOV1-LOV2 double domains is compared with the behaviour of the separate LOV1 and LOV2 domains.

Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

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Bernardas Morkunas

2016-07-01

Full Text Available Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

Decreased spontaneous activity in AMPK alpha 2 muscle specific kinase dead mice is not caused by changes in brain dopamine metabolism

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Møller, Lisbeth Liliendal Valbjørn; Sylow, Lykke; Gøtzsche, Casper René

2016-01-01

was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19 days. AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared...... through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running...... capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity...

TDRP deficiency contributes to low sperm motility and is a potential risk factor for male infertility.

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Mao, Shanhua; Wu, Fei; Cao, Xinyi; He, Min; Liu, Naijia; Wu, Huihui; Yang, Zhihong; Ding, Qiang; Wang, Xuanchun

2016-01-01

TDRP (Testis Development-Related Protein), a nuclear factor, might play an important role in spermatogenesis. However, the molecular mechanisms of TDRP underlying these fundamental processes remain elusive. In this study, a Tdrp-deficient mouse model was generated. Fertility tests and semen analysis were performed. Tdrp-deficient mice were not significantly different from wild-type littermates in development of testes, genitourinary tract, or sperm count. Morphologically, spermatozoa of the Tdrp-deficient mice was not significantly different from the wild type. Several sperm motility indexes, i.e. the average path velocity (VAP), the straight line velocity (VSL) and the curvilinear velocity (VCL) were significantly decreased in Tdrp-deficient mice (psperm also increased significantly in the mutant mice (psperm motility, but Tdrp deficiency alone was not sufficient to cause male infertility in mice. Additionally, TDRP1 might participate in spermatogenes is through interaction with PRM2.

The Role of Fibroblast Growth Factor Binding Protein 1 in Skin Carcinogenesis and Inflammation

DEFF Research Database (Denmark)

Schmidt, Marcel Oliver; Garman, Khalid Ammar; Lee, Yong Gu

2018-01-01

, and is upregulated in various cancers. Here we evaluated the contribution of endogenous FGFBP1 to development and homeostasis as well as to skin pathologies utilizing Fgfbp1-knockout (KO) mice. Relative to wild-type (WT) littermates KO mice showed no gross pathologies. Still, in KO mice a significant thickening...... of the epidermis associated with a decreased transepidermal water loss and increased pro-inflammatory gene expression in the skin was detected. Also, skin carcinogen challenge by DMBA/TPA resulted in delayed and reduced papillomatosis in KO mice. This was paralleled by delayed healing of skin wounds and reduced...... angiogenic sprouting in subcutaneous matrigel plugs. Heterozygous GFP-knock-in mice revealed rapid induction of gene expression during papilloma induction and during wound healing. Examination of WT skin grafted onto Fgfbp1 GFP knockin reporter hosts and bone marrow transplants from the GFP reporter model...

Role of adenosine 5'-monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle

DEFF Research Database (Denmark)

Glund, Stephan; Treebak, Jonas Thue; Long, Yun Chau

2009-01-01

IL-6 is released from skeletal muscle during exercise and has consequently been implicated to mediate beneficial effects on whole-body metabolism. Using 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), a pharmacological activator of 5'-AMP-activated protein kinase (AMPK), we tested......-type mice was also incubated with the AMPK activator A-769662. Incubation of mouse glycolytic extensor digitorum longus and oxidative soleus muscle for 2 h was associated with profound IL-6 mRNA production and protein release, which was suppressed by AICAR (P ... the hypothesis that AMPK modulates IL-6 release from isolated muscle. Skeletal muscle from AMPKalpha2 kinase-dead transgenic, AMPKalpha1 knockout (KO) and AMPKgamma3 KO mice and respective wild-type littermates was incubated in vitro, in the absence or presence of 2 mmol/liter AICAR. Skeletal muscle from wild...

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

DEFF Research Database (Denmark)

Maccarana, M.; Kalamajski, S.; Kongsgaard, M.

2009-01-01

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks...... of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican......-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found...

Exploration, anxiety, and spatial memory in transgenic anophthalmic mice.

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Buhot, M C; Dubayle, D; Malleret, G; Javerzat, S; Segu, L

2001-04-01

Contradictory results are found in the literature concerning the role of vision in the perception of space or in spatial navigation, in part because of the lack of murine models of total blindness used so far. The authors evaluated the spatial abilities of anophthalmic transgenic mice. These mice did not differ qualitatively from their wild-type littermates in general locomotor activity, spontaneous alternation, object exploration, or anxiety, but their level of exploratory activity was generally lower. In the spatial version of the water maze, they displayed persistent thigmotaxic behavior and showed severe spatial learning impairments. However, their performances improved with training, suggesting that they may have acquired a rough representation of the platform position. These results suggest that modalities other than vision enable some degree of spatial processing in proximal and structured spaces but that vision is critical for accurate spatial navigation.

The RabGAP TBC1D1 plays a central role in exercise-regulated glucose metabolism in skeletal muscle

DEFF Research Database (Denmark)

Stöckli, Jacqueline; Meoli, Christopher C; Hoffman, Nolan J

2015-01-01

Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated...... weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance...... together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers....

Effects of ketoconazole on the biodistribution and metabolism of [{sup 11}C]loperamide and [{sup 11}C]N-desmethyl-loperamide in wild-type and P-gp knockout mice

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Seneca, Nicholas; Zoghbi, Sami S.; Shetty, H. Umesha; Tuan, Edward; Kannan, Pavitra; Taku, Andrew; Innis, Robert B. [Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 (United States); Pike, Victor W. [Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: pikev@mail.nih.gov

2010-04-15

Introduction: [{sup 11}C]Loperamide and [{sup 11}C]N-desmethyl-loperamide ([{sup 11}C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [{sup 11}C]loperamide metabolism is N-demethylation to [{sup 11}C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [{sup 11}C]loperamide and [{sup 11}C]dLop in mice, and thereby improve the quality of these radiotracers. Methods: Studies were performed in wild-type and P-gp knockout (mdr-1a/b -/-) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, was pretreated with ketoconazole (50 mg/kg, ip), while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [{sup 11}C]loperamide or [{sup 11}C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-high-performance liquid chromatography. Results: Ketoconazole increased the plasma concentrations of [{sup 11}C]loperamide and its main radiometabolite, [{sup 11}C]dLop, by about twofold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased threefold. Furthermore, ketoconazole increased the brain concentrations of [{sup 11}C]loperamide and the radiometabolite [{sup 11}C]dLop by about twofold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82% and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [{sup 11}C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [{sup 11}C]dLop concentration. The least polar radiometabolite of [{sup 11}C]dLop was identified with LC-MS{sup n} as the N-hydroxymethyl analog of [{sup 11}C]dLop and this also behaved as a P-gp substrate. Conclusion: In this study, ketoconazole (50 mg/kg, ip) proved

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

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Yamaura, Takumi; Ezaki, Junji; Okabe, Naoyuki; Takagi, Hironori; Ozaki, Yuki; Inoue, Takuya; Watanabe, Yuzuru; Fukuhara, Mitsuro; Muto, Satoshi; Matsumura, Yuki; Hasegawa, Takeo; Hoshino, Mika; Osugi, Jun; Shio, Yutaka; Waguri, Satoshi; Tamura, Hirosumi; Imai, Jun-Ichi; Ito, Emi; Yanagisawa, Yuka; Honma, Reiko; Watanabe, Shinya; Suzuki, Hiroyuki

2018-02-01

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor ( EGFR ) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B ( FAM83B ) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR . Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR . Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR .

Chronic mild stress impairs latent inhibition and induces region-specific neural activation in CHL1-deficient mice, a mouse model of schizophrenia.

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Buhusi, Mona; Obray, Daniel; Guercio, Bret; Bartlett, Mitchell J; Buhusi, Catalin V

2017-08-30

Schizophrenia is a neurodevelopmental disorder characterized by abnormal processing of information and attentional deficits. Schizophrenia has a high genetic component but is precipitated by environmental factors, as proposed by the 'two-hit' theory of schizophrenia. Here we compared latent inhibition as a measure of learning and attention, in CHL1-deficient mice, an animal model of schizophrenia, and their wild-type littermates, under no-stress and chronic mild stress conditions. All unstressed mice as well as the stressed wild-type mice showed latent inhibition. In contrast, CHL1-deficient mice did not show latent inhibition after exposure to chronic stress. Differences in neuronal activation (c-Fos-positive cell counts) were noted in brain regions associated with latent inhibition: Neuronal activation in the prelimbic/infralimbic cortices and the nucleus accumbens shell was affected solely by stress. Neuronal activation in basolateral amygdala and ventral hippocampus was affected independently by stress and genotype. Most importantly, neural activation in nucleus accumbens core was affected by the interaction between stress and genotype. These results provide strong support for a 'two-hit' (genes x environment) effect on latent inhibition in CHL1-deficient mice, and identify CHL1-deficient mice as a model of schizophrenia-like learning and attention impairments. Copyright © 2017 Elsevier B.V. All rights reserved.

NMDA and kainate receptor expression, long-term potentiation, and neurogenesis in the hippocampus of long-lived Ames dwarf mice.

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Sharma, Sunita; Darland, Diane; Lei, Saobo; Rakoczy, Sharlene; Brown-Borg, Holly M

2012-06-01

In the current study, we investigated changes in N-methyl D-aspartate (NMDA) and kainate receptor expression, long-term potentiation (LTP), and neurogenesis in response to neurotoxic stress in long-living Ames dwarf mice. We hypothesized that Ames dwarf mice have enhanced neurogenesis that enables retention of spatial learning and memory with age and promotes neurogenesis in response to injury. Levels of the NMDA receptors (NR)1, NR2A, NR2B, and the kainate receptor (KAR)2 were increased in Ames dwarf mice, relative to wild-type littermates. Quantitative assessment of the excitatory postsynaptic potential in Schaffer collaterals in hippocampal slices from Ames dwarf mice showed an increased response in high-frequency induced LTP over time compared with wild type. Kainic acid (KA) injection was used to promote neurotoxic stress-induced neurogenesis. KA mildly increased the number of doublecortin-positive neurons in wild-type mice, but the response was significantly enhanced in the Ames dwarf mice. Collectively, these data support our hypothesis that the enhanced learning and memory associated with the Ames dwarf mouse may be due to elevated levels of NMDA and KA receptors in hippocampus and their ability to continue producing new neurons in response to neuronal damage.

Species-relevant inescapable stress differently influences memory consolidation and retrieval of mice in a spatial radial arm maze.

Science.gov (United States)

Janitzky, K; Schwegler, H; Kröber, A; Roskoden, T; Yanagawa, Y; Linke, R

2011-05-16

Stress affects learning and there are both facilitating and impairing actions of stressors on memory processes. Here we investigated the influence of acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), an ethological relevant stressor for rodents, on spatial memory formation and performance in a radial arm maze (RAM) task and studied TMT effects on corticosterone levels in GAD67-GFP knock-in mice and their wildtype littermates. Our results suggest that predator odor-exposure differently affects consolidation and retrieval of memory in a hippocampus-dependent spatial learning task in adult male mice, independently from their genotypes. Acute TMT-stress before retrieval facilitates performance, whereas repeated TMT-stress during consolidation exerts no influence. Additionally, we found genotype specific effects of TMT on corticosterone release. While TMT-stress tend to result in increased corticosterone release in wildtypes there was a significant decrease in transgenic mice. Taken together, these findings indicate that biologically significant predator odor-induced stress can have different actions on the strength of spatial memory formation depending on the timing with regard to memory phases. Furthermore, we suppose an impact of GABAergic mechanisms on HPA-stress axis activation to TMT resulting in absent peripheral corticosterone release of GAD67-GFP mice. Copyright © 2011 Elsevier B.V. All rights reserved.

Shorter duration of non-rapid eye movement sleep slow waves in EphA4 knockout mice.

Science.gov (United States)

Freyburger, Marlène; Poirier, Gaétan; Carrier, Julie; Mongrain, Valérie

2017-10-01

Slow waves occurring during non-rapid eye movement sleep have been associated with neurobehavioural performance and memory. In addition, the duration of previous wakefulness and sleep impacts characteristics of these slow waves. However, molecular mechanisms regulating the dynamics of slow-wave characteristics remain poorly understood. The EphA4 receptor regulates glutamatergic transmission and synaptic plasticity, which have both been linked to sleep slow waves. To investigate if EphA4 regulates slow-wave characteristics during non-rapid eye movement sleep, we compared individual parameters of slow waves between EphA4 knockout mice and wild-type littermates under baseline conditions and after a 6-h sleep deprivation. We observed that, compared with wild-type mice, knockout mice display a shorter duration of positive and negative phases of slow waves under baseline conditions and after sleep deprivation. However, the mutation did not change slow-wave density, amplitude and slope, and did not affect the sleep deprivation-dependent changes in slow-wave characteristics, suggesting that EphA4 is not involved in the response to elevated sleep pressure. Our present findings suggest a role for EphA4 in shaping cortical oscillations during sleep that is independent from sleep need. © 2017 European Sleep Research Society.

Comparison of anti-inflammatory activity of extracts with supercritical carbon dioxide from radiation mutant perilla frutescens(L.) Britton and wild-type

Energy Technology Data Exchange (ETDEWEB)

Park, Han Chul; So, Yang Kang; Kim, Jin Baek; Jin, Chang Hyun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Yuk, Hong Sun [Dept. of Food and Nutrition, Chungnam National University Daejeon (Korea, Republic of)

2016-11-15

In previous study, the radiation mutant Perilla frutescens (L.) Britton with a higher anti-inflammatory activity was selected. The extracts were obtained from the mutant and wildtype using a supercritical carbon dioxide technique. This study aimed to compare the antiinflammatory activities between the mutant supercritical extract (MSE) and wild-type supercritical extract (WSE). The contents of isoegomaketone (IK) of MSE and WSE were measured through an HPLC analysis. MSE contained IK contents approximately 7-fold higher than those of WSE. To compare the anti-inflammatory activities of MSE and WSE, the expression levels of the mRNA and protein of pro-inflammatory mediators were measured in lipopolysaccharide (LPS)-induced RAW264.7 cells. As a result, MSE inhibited the expression levels of the mRNA and protein of pro-inflammatory mediators, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) to a much greater extent than did WSE. Taken together, MSE had more IK contents and higher antiinflammatory activities than WSE. Therefore, MSE is proposed based on its therapeutic potential in the prevention of inflammatory disease.

Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

Science.gov (United States)

Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

2017-11-01

Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

iTRAQ-facilitated proteomic profiling of anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.).

Science.gov (United States)

Liu, Ji; Pang, Chaoyou; Wei, Hengling; Song, Meizhen; Meng, Yanyan; Ma, Jianhui; Fan, Shuli; Yu, Shuxun

2015-08-03

Male sterility is a common phenomenon in flowering plants, and it has been successfully developed in several crops by taking advantage of heterosis. Cotton (Gossypium hirsutum L.) is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To use CCRI9106 in cotton hybrid breeding, it is of great importance to study the molecular mechanisms of its male sterility. Here, histological and iTRAQ-facilitated proteomic analyses of anthers were performed to explore male sterility mechanisms of the mutant. Scanning and transmission electron microscopy of the anthers showed that the development of pollen wall in CCRI9106 was severely defective with a lack of exine formation. At the protein level, 6121 high-confidence proteins were identified and 325 of them showed differential expression patterns between mutant and wild-type anthers. The proteins up- or down-regulated in MT anthers were mainly involved in exine formation, protein degradation, calcium ion binding,etc. These findings provide valuable information on the proteins involved in anther and pollen development, and contribute to elucidate the mechanism of male sterility in upland cotton. Copyright © 2015. Published by Elsevier B.V.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics

Science.gov (United States)

Wong, Min Hao; Giraldo, Juan P.; Kwak, Seon-Yeong; Koman, Volodymyr B.; Sinclair, Rosalie; Lew, Tedrick Thomas Salim; Bisker, Gili; Liu, Pingwei; Strano, Michael S.

2017-02-01

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors--single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal--embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome.

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Onetti, Yara; Meirelles, Thayna; Dantas, Ana P; Schröder, Katrin; Vila, Elisabet; Egea, Gustavo; Jiménez-Altayó, Francesc

2016-05-01

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS. Copyright © 2016 the American Physiological Society.

Knockdown of angiopoietin-like 2 mimics the benefits of intermittent fasting on insulin responsiveness and weight loss.

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Martel, Cécile; Pinçon, Anthony; Bélanger, Alexandre Maxime; Luo, Xiaoyan; Gillis, Marc-Antoine; de Montgolfier, Olivia; Thorin-Trescases, Nathalie; Thorin, Éric

2018-01-01

Angiopoietin-like 2 (ANGPTL2) is an inflammatory adipokine linking obesity to insulin resistance. Intermittent fasting, on the other hand, is a lifestyle intervention able to prevent obesity and diabetes but difficult to implement and maintain. Our objectives were to characterize a link between ANGPTL2 and intermittent fasting and to investigate whether the knockdown of ANGPTL2 reproduces the benefits of intermittent fasting on weight gain and insulin responsiveness in knockdown and wild-type littermates mice. Intermittent fasting, access to food ad libitum once every other day, was initiated at the age of three months and maintained for four months. Intermittent fasting decreased by 63% (p < 0.05) gene expression of angptl2 in adipose tissue of wild-type mice. As expected, intermittent fasting improved insulin sensitivity (p < 0.05) and limited weight gain (p < 0.05) in wild-type mice. Knockdown mice fed ad libitum, however, were comparable to wild-type mice following the intermittent fasting regimen: insulin sensitivity and weight gain were identical, while intermittent fasting had no additional impact on these parameters in knockdown mice. Energy intake was similar between both wild-type fed intermittent fasting and ANGPTL2 knockdown mice fed ad libitum, suggesting that intermittent fasting and knockdown of ANGPTL2 equally lower feeding efficiency. These results suggest that the reduction of ANGPTL2 could be a useful and promising strategy to prevent obesity and insulin resistance, although further investigation of the mechanisms linking ANGPTL2 and intermittent fasting is warranted. Impact statement Intermittent fasting is an efficient diet pattern to prevent weight gain and improve insulin sensitivity. It is, however, a difficult regimen to follow and compliance is expected to be very low. In this work, we demonstrate that knockdown of ANGPTL2 in mice fed ad libitum mimics the beneficial effects of intermittent fasting on weight gain and insulin

Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

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Xinhe Wang

2015-07-01

Full Text Available The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p. inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) — Differences between vaccinated and wild-type virus exposed dogs

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Perrone, Danielle; Bender, Scott; Niewiesk, Stefan

2010-01-01

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals. PMID:20885846

cGAS-mediated control of blood-stage malaria promotes Plasmodium-specific germinal center responses.

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Hahn, William O; Butler, Noah S; Lindner, Scott E; Akilesh, Holly M; Sather, D Noah; Kappe, Stefan Hi; Hamerman, Jessica A; Gale, Michael; Liles, W Conrad; Pepper, Marion

2018-01-25

Sensing of pathogens by host pattern recognition receptors is essential for activating the immune response during infection. We used a nonlethal murine model of malaria (Plasmodium yoelii 17XNL) to assess the contribution of the pattern recognition receptor cyclic GMP-AMP synthase (cGAS) to the development of humoral immunity. Despite previous reports suggesting a critical, intrinsic role for cGAS in early B cell responses, cGAS-deficient (cGAS-/-) mice had no defect in the early expansion or differentiation of Plasmodium-specific B cells. As the infection proceeded, however, cGAS-/- mice exhibited higher parasite burdens and aberrant germinal center and memory B cell formation when compared with littermate controls. Antimalarial drugs were used to further demonstrate that the disrupted humoral response was not B cell intrinsic but instead was a secondary effect of a loss of parasite control. These findings therefore demonstrate that cGAS-mediated innate-sensing contributes to parasite control but is not intrinsically required for the development of humoral immunity. Our findings highlight the need to consider the indirect effects of pathogen burden in investigations examining how the innate immune system affects the adaptive immune response.

Deficiency of vasodilator-stimulated phosphoprotein (VASP increases blood-brain-barrier damage and edema formation after ischemic stroke in mice.

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Peter Kraft

2010-12-01

Full Text Available Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification.Focal cerebral ischemia was induced in Vasp(-/- mice and wild-type (WT littermates by transient middle cerebral artery occlusion (tMCAO. Evan's Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p0.05 towards worse neurological outcomes.Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.

Impaired spatial processing in a mouse model of fragile X syndrome.

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Ghilan, Mohamed; Bettio, Luis E B; Noonan, Athena; Brocardo, Patricia S; Gil-Mohapel, Joana; Christie, Brian R

2018-05-17

Fragile X syndrome (FXS) is the most common form of inherited intellectual impairment. The Fmr1 -/y mouse model has been previously shown to have deficits in context discrimination tasks but not in the elevated plus-maze. To further characterize this FXS mouse model and determine whether hippocampal-mediated behaviours are affected in these mice, dentate gyrus (DG)-dependent spatial processing and Cornu ammonis 1 (CA1)-dependent temporal order discrimination tasks were evaluated. In agreement with previous findings of long-term potentiation deficits in the DG of this transgenic model of FXS, the results reported here demonstrate that Fmr1 -/y mice perform poorly in the DG-dependent metric change spatial processing task. However, Fmr1 -/y mice did not present deficits in the CA1-dependent temporal order discrimination task, and were able to remember the order in which objects were presented to them to the same extent as their wild-type littermate controls. These data suggest that the previously reported subregional-specific differences in hippocampal synaptic plasticity observed in the Fmr1 -/y mouse model may manifest as selective behavioural deficits in hippocampal-dependent tasks. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Relative Tissue Factor Deficiency Attenuates Ventilator-Induced Coagulopathy but Does Not Protect against Ventilator-Induced Lung Injury in Mice

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Esther K. Wolthuis

2012-01-01

Full Text Available Preventing tissue-factor-(TF- mediated systemic coagulopathy improves outcome in models of sepsis. Preventing TF-mediated pulmonary coagulopathy could attenuate ventilator-induced lung injury (VILI. We investigated the effect of relative TF deficiency on pulmonary coagulopathy and inflammation in a murine model of VILI. Heterozygous TF knockout (TF+/− mice and their wild-type (TF+/+ littermates were sedated (controls or sedated, tracheotomized, and mechanically ventilated with either low or high tidal volumes for 5 hours. Mechanical ventilation resulted in pulmonary coagulopathy and inflammation, with more injury after mechanical ventilation with higher tidal volumes. Compared with TF+/+ mice, TF+/− mice demonstrated significantly lower pulmonary thrombin-antithrombin complex levels in both ventilation groups. There were, however, no differences in lung wet-to-dry ratio, BALF total protein levels, neutrophil influx, and lung histopathology scores between TF+/− and TF+/+ mice. Notably, pulmonary levels of cytokines were significantly higher in TF+/− as compared to TF+/+ mice. Systemic levels of cytokines were not altered by the relative absence of TF. TF deficiency is associated with decreased pulmonary coagulation independent of the ventilation strategy. However, relative TF deficiency does not reduce VILI and actually results in higher pulmonary levels of inflammatory mediators.

Depot-Specific Response of Adipose Tissue to Diet-Induced Inflammation: The Retinoid-Related Orphan Receptor α (RORα) Involved?

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Kadiri, Sarah; Auclair, Martine; Capeau, Jacqueline; Antoine, Bénédicte

2017-11-01

Epididymal adipose tissue (EAT), a visceral fat depot, is more closely associated with metabolic dysfunction than inguinal adipose tissue (IAT), a subcutaneous depot. This study evaluated whether the nuclear receptor RORα, which controls inflammatory processes, could be implicated. EAT and IAT were compared in a RORα loss-of-function mouse (sg/sg) and in wild-type (WT) littermates, fed a standard diet (SD) or a Western diet (WD), to evaluate the impact of RORα expression on inflammatory status and on insulin sensitivity (IS) of each fat depot according to the diet. Sg/sg mice fed the SD exhibited a decreased inflammatory status and a higher IS in their fat depots than WT mice. WD-induced obesity had distinct effects on the two fat depots. In WT mice, EAT exhibited increased inflammation and insulin resistance while IAT showed reduced inflammation and improved IS, together with a depot-specific increase of RORα, and its target gene IκBα, in the stroma vascular fraction (SVF). Conversely, in sg/sg mice, WD increased inflammation and lowered IS of IAT but not of EAT. These findings suggest an anti-inflammatory role for RORα in response to WD, which occurs at the level of SVF of IAT, thus possibly contributing to the "healthy" expansion of IAT. © 2017 The Obesity Society.

Quantification of heterogeneity in lung disease with image-based pulmonary function testing.

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Stahr, Charlene S; Samarage, Chaminda R; Donnelley, Martin; Farrow, Nigel; Morgan, Kaye S; Zosky, Graeme; Boucher, Richard C; Siu, Karen K W; Mall, Marcus A; Parsons, David W; Dubsky, Stephen; Fouras, Andreas

2016-07-27

Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease, and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children, and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in β-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in β-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction, and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.

Overload-mediated skeletal muscle hypertrophy is not impaired by loss of myofiber STAT3.

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Pérez-Schindler, Joaquín; Esparza, Mary C; McKendry, James; Breen, Leigh; Philp, Andrew; Schenk, Simon

2017-09-01

Although the signal pathways mediating muscle protein synthesis and degradation are well characterized, the transcriptional processes modulating skeletal muscle mass and adaptive growth are poorly understood. Recently, studies in mouse models of muscle wasting or acutely exercised human muscle have suggested a potential role for the transcription factor signal transducer and activator of transcription 3 (STAT3), in adaptive growth. Hence, in the present study we sought to define the contribution of STAT3 to skeletal muscle adaptive growth. In contrast to previous work, two different resistance exercise protocols did not change STAT3 phosphorylation in human skeletal muscle. To directly address the role of STAT3 in load-induced (i.e., adaptive) growth, we studied the anabolic effects of 14 days of synergist ablation (SA) in skeletal muscle-specific STAT3 knockout (mKO) mice and their floxed, wild-type (WT) littermates. Plantaris muscle weight and fiber area in the nonoperated leg (control; CON) was comparable between genotypes. As expected, SA significantly increased plantaris weight, muscle fiber cross-sectional area, and anabolic signaling in WT mice, although interestingly, this induction was not impaired in STAT3 mKO mice. Collectively, these data demonstrate that STAT3 is not required for overload-mediated hypertrophy in mouse skeletal muscle. Copyright © 2017 the American Physiological Society.

Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation.

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Outram, Susan V; Hager-Theodorides, Ariadne L; Shah, Divya K; Rowbotham, Nicola J; Drakopoulou, Ekati; Ross, Susan E; Lanske, Beate; Dessens, Johannes T; Crompton, Tessa

2009-03-05

We show that Indian Hedgehog (Ihh) regulates T-cell development and homeostasis in both fetal and adult thymus, controlling thymocyte number. Fetal Ihh(-/-) thymi had reduced differentiation to double-positive (DP) cell and reduced cell numbers compared with wild-type littermates. Surprisingly, fetal Ihh(+/-) thymi had increased thymocyte numbers and proportion of DP cells relative to wild type, indicating that Ihh also negatively regulates thymocyte development. In vitro treatment of thymus explants with exogenous recombinant Hedgehog protein promoted thymocyte development in Ihh(-/-) thymi but inhibited thymocyte development in Ihh(+/-), confirming both positive and negative regulatory functions of Ihh. Analysis of Rag(-/-)Ihh(+/-) thymi showed that Ihh promotes T-cell development before pre-T-cell receptor (pre-TCR) signaling, but negatively regulates T-cell development only after pre-TCR signaling has taken place. We show that Ihh is most highly expressed by the DP population and that Ihh produced by DP cells feeds back to negatively regulate the differentiation and proliferation of their double-negative progenitors. Thus, differentiation from double-negative to DP cell, and hence the size of the DP population, is dependent on the concentration of Ihh in the thymus. Analysis of Ihh conditional knockout and heterozygote adult mice showed that Ihh also influences thymocyte number in the adult.

Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease.

OpenAIRE

Franklin R Toapanta; Paula J Bernal; Stephanie Fresnay; Laurence S Magder; Thomas C Darton; Claire Jones; Claire S Waddington; Christoph J Blohmke; Brian Angus; Myron M Levine; Andrew J Pollard; Marcelo B Sztein

2016-01-01

A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD-) 6?9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ?38?C for ?12h) and/or microbiological (S. Typhi bacteremia) endpoints. Changes in B cell subpopulations following S. Typhi challenge re...

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

Science.gov (United States)

Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8�

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data.

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Angeby, K A; Jureen, P; Giske, C G; Chryssanthou, E; Sturegård, E; Nordvall, M; Johansson, A G; Werngren, J; Kahlmeter, G; Hoffner, S E; Schön, T

2010-05-01

To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. We propose S (susceptible)

Cetuximab Plus Various Chemotherapy Regimens for Patients with KRAS Wild-Type Metastatic Colorectal Cancer.

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Azadeh, Payam; Mortazavi, Nafiseh; Tahmasebi, Arezoo; Hosseini Kamal, Farnaz; Novin, Kambiz

2016-01-01

The aim of this study was to compare the efficacy and hematologic toxicity of cetuximab combined with various types of chemotherapy regimens in patients with KRAS wild-type metastatic colorectal cancer (mCRC). The response rate, progression-free survival (PFS) and overall survival of the patients were analyzed. In total, 45 patients were included in the study. The overall response rate for the combination of cetuximab and FOLFOX, FOLFIRI and CAPOX was 20, 46 and 30%, respectively, but the differences were not statistically significant. The median PFS for the three groups were 8, 6 and 3.5 months, respectively, but again these differences were not significant. All-grade leukopenia and anemia for the cetuximab plus FOLFOX group were significantly higher than for the other chemotherapy regimens. Our findings suggest that the combination of cetuximab and the three standard chemotherapy regimens resulted in the same outcomes in our patient population of mCRC, with higher hematologic toxicities among the FOLFOX subgroup. © 2015 S. Karger AG, Basel.

Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

International Nuclear Information System (INIS)

Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

1987-01-01

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

Nrf2 but not autophagy inhibition is associated with the survival of wild-type epidermal growth factor receptor non-small cell lung cancer cells

International Nuclear Information System (INIS)

Zhou, Yan; Li, Yuan; Ni, Hong-Min; Ding, Wen-Xing; Zhong, Hua

2016-01-01

Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the world. Icotinib and Gefitinib are two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that have been used to treat NSCLC. While it is well known that mutations of EGFR can affect the sensitivity of NSCLC to the EGFR-TKI, other mechanisms may also be adopted by lung cancer cells to develop resistance to EGFR-TKI treatment. Cancer cells can use multiple adaptive mechanisms such as activation of autophagy and Nrf2 to protect against various stresses and chemotherapeutic drugs. Whether autophagy or Nrf2 activation contributes to the resistance of NSCLC to EGFR-TKI treatment in wild-type EGFR NSCLC cells remains elusive. In the present study, we confirmed that Icotinib and Gefitinib induced apoptosis in EGFR mutant HCC827 but not in EGFR wild-type A549 NSCLC cells. Icotinib and Gefitinib did not induce autophagic flux or inhibit mTOR in A549 cells. Moreover, suppression of autophagy by chloroquine, a lysosomal inhibitor, did not affect Icotinib- or Gefitinib-induced cell death in A549 cells. In contrast, Brusatol, an Nrf2 inhibitor, significantly suppressed the cell survival of A549 cells. However, Brusatol did not further sensitize A549 cells to EGFR TKI-induced cell death. Results from this study suggest that inhibition of Nrf2 can decrease cell vitality of EGFR wild-type A549 cells independent of autophagy. - Highlights: • Cancer cells use adaptive mechanisms against chemotherapy. • Autophagy is not essential for the drug resistance of lung cancer A549 cells. • Inhibition of Nrf2 decreases cell survival of lung cancer A549 cells.

Nrf2 but not autophagy inhibition is associated with the survival of wild-type epidermal growth factor receptor non-small cell lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Zhou, Yan [Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030 (China); Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160 (United States); Li, Yuan; Ni, Hong-Min; Ding, Wen-Xing [Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160 (United States); Zhong, Hua, E-mail: eddiedong8@hotmail.com [Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030 (China)

2016-11-01

Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the world. Icotinib and Gefitinib are two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that have been used to treat NSCLC. While it is well known that mutations of EGFR can affect the sensitivity of NSCLC to the EGFR-TKI, other mechanisms may also be adopted by lung cancer cells to develop resistance to EGFR-TKI treatment. Cancer cells can use multiple adaptive mechanisms such as activation of autophagy and Nrf2 to protect against various stresses and chemotherapeutic drugs. Whether autophagy or Nrf2 activation contributes to the resistance of NSCLC to EGFR-TKI treatment in wild-type EGFR NSCLC cells remains elusive. In the present study, we confirmed that Icotinib and Gefitinib induced apoptosis in EGFR mutant HCC827 but not in EGFR wild-type A549 NSCLC cells. Icotinib and Gefitinib did not induce autophagic flux or inhibit mTOR in A549 cells. Moreover, suppression of autophagy by chloroquine, a lysosomal inhibitor, did not affect Icotinib- or Gefitinib-induced cell death in A549 cells. In contrast, Brusatol, an Nrf2 inhibitor, significantly suppressed the cell survival of A549 cells. However, Brusatol did not further sensitize A549 cells to EGFR TKI-induced cell death. Results from this study suggest that inhibition of Nrf2 can decrease cell vitality of EGFR wild-type A549 cells independent of autophagy. - Highlights: • Cancer cells use adaptive mechanisms against chemotherapy. • Autophagy is not essential for the drug resistance of lung cancer A549 cells. • Inhibition of Nrf2 decreases cell survival of lung cancer A549 cells.

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

Science.gov (United States)

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

2016-09-13

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

Science.gov (United States)

Song, Xiaoyun; Liu, Xingcai; Ding, Xi

2017-04-01

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.

OpenAIRE

Cox, D. G.; Simard, J.; Sinnett, D.; Hamdi, Y.; Soucy, P.; Ouimet, M.; Barjhoux, L.; Verny-Pierre, C.; McGuffog, L.; Healey, S.; Szabo, C.; Greene, M. H.; Mai, P. L.; Andrulis, I. L.; Thomassen, M.

2011-01-01

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA...

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Fibroblast growth factor 21 is required for beneficial effects of exercise during chronic high-fat feeding

Science.gov (United States)

Loyd, Christine; Magrisso, I. Jack; Haas, Michael; Balusu, Sowmya; Krishna, Radha; Itoh, Nobuyuki; Sandoval, Darleen A.; Perez-Tilve, Diego; Obici, Silvana

2016-01-01

Exercise is an effective therapy against the metabolic syndrome. However, the molecular pathways underlying the advantageous effects of exercise are elusive. Glucagon receptor signaling is essential for exercise benefits, and recent evidence indicates that a downstream effector of glucagon, fibroblast growth factor 21 (FGF21), is implicated in this response. Therefore, we tested the hypothesis that FGF21 action is necessary in mediating metabolic effects of exercise. We utilized acute exhaustive treadmill exercise in Wistar rats to identify a putative, concomitant increase in plasma glucagon and FGF21 with the increase in glucose and lactate following exercise. To test the necessity of FGF21 action in the exercise response, we exposed FGF21 congenitally deficient mice (Fgf21−/−) and their wild-type (Wt) littermates to chronic high-fat (HF) feeding and inoperable (sedentary) or operable (exercise) voluntary running wheels. Physiological tests were performed to assess the role of FGF21 in the beneficial effect of exercise on glucose metabolism. Wt and Fgf21−/− littermates exhibited similar running behavior, and exercise was effective in suppressing weight and fat mass gain and dyslipidemia independently of genotype. However, exercise failed to positively affect hepatic triglyceride content and glucose tolerance in HF diet-fed Fgf21−/− mice. Furthermore, Fgf21−/− mice exhibited an impaired adaptation to exercise training, including reduced AMP-activated protein kinase activity in skeletal muscle. This study demonstrates that FGF21 action is necessary to achieve the full metabolic benefits of exercise during chronic HF feeding. PMID:27445299

Tight Skin 2 Mice Exhibit Delayed Wound Healing Caused by Increased Elastic Fibers in Fibrotic Skin.

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Long, Kristen B; Burgwin, Chelsea M; Huneke, Richard; Artlett, Carol M; Blankenhorn, Elizabeth P

2014-09-01

Rationale: The Tight Skin 2 (Tsk2) mouse model of systemic sclerosis (SSc) has many features of human disease, including tight skin, excessive collagen deposition, alterations in the extracellular matrix (ECM), increased elastic fibers, and occurrence of antinuclear antibodies with age. A tight skin phenotype is observed by 2 weeks of age, but measurable skin fibrosis is only apparent at 10 weeks. We completed a series of wound healing experiments to determine how fibrosis affects wound healing in Tsk2/+ mice compared with their wild-type (WT) littermates. Method: We performed these experiments by introducing four 4 mm biopsy punched wounds on the back of each mouse, ventral of the midline, and observed wound healing over 10 days. Tsk2/+ mice showed significantly delayed wound healing and increased wound size compared with the WT littermates at both 5 and 10 weeks of age. We explored the potential sources of this response by wounding Tsk2/+ mice that were genetically deficient either for the NLRP3 inflammasome (a known fibrosis mediator), or for elastic fibers in the skin, using a fibulin-5 knockout. Conclusion: We found that the loss of elastic fibers restores normal wound healing in the Tsk2/+ mouse and that the loss of the NLRP3 inflammasome had no effect. We conclude that elastic fiber dysregulation is the primary cause of delayed wound healing in the Tsk2/+ mouse and therapies that promote collagen deposition in the tissue matrix in the absence of elastin deposition might be beneficial in promoting wound healing in SSc and other diseases.

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

International Nuclear Information System (INIS)

Hu, Bo; He, Xiusheng; Li, Ao; Qiu, Qingchao; Li, Cunxi; Liang, Dan; Zhao, Ping; Ma, Jie; Coffey, Robert J.; Zhan, Qimin; Wu, Guanqing

2011-01-01

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1 -/- renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1 -/- cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1 -/- mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1 -/- kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism.

Science.gov (United States)

Ju, Anes; Hammerschmidt, Kurt; Tantra, Martesa; Krueger, Dilja; Brose, Nils; Ehrenreich, Hannelore

2014-08-15

Neuroligin-4 (Nlgn4) is a member of the neuroligin family of postsynaptic cell adhesion molecules. Loss-of-function mutations of NLGN4 are among the most frequent, known genetic causes of heritable autism. Adult Nlgn4 null mutant (Nlgn4(-/-)) mice are a construct valid model of human autism, with both genders displaying a remarkable autistic phenotype, including deficits in social interaction and communication as well as restricted and repetitive behaviors. In contrast to adults, autism-related abnormalities in neonatal and juvenile Nlgn4(-/-) mice have not been reported yet. The present study has been designed to systematically investigate in male and female Nlgn4(-/-) pups versus wildtype littermates (WT, Nlgn4(+/+)) developmental milestones and stimulus-induced ultrasound vocalization (USV). Neonatal development, followed daily from postnatal days (PND) 4 to 21, including physical development, neurological reflexes and neuromotor coordination, did not yield any differences between Nlgn4(-/-) and their WT littermates. USV in pups (PND8-9) in response to brief separation from their mothers revealed remarkable gender effects, and a genotype influence in females regarding latency to first call. In juveniles (PND22-23), USV monitoring upon exposure to an anesthetized female intruder mouse uncovered a clear genotype effect with reduced USV in Nlgn4(-/-) mice, and again a more prominent phenotype in females. Together, these data support an early manifestation of communication deficits in Nlgn4(-/-) mice that appear more pronounced in immature females with their overall stronger USV as compared to males. Copyright © 2014 Elsevier B.V. All rights reserved.

Glycomic analyses of mouse models of congenital muscular dystrophy.

Science.gov (United States)

Stalnaker, Stephanie H; Aoki, Kazuhiro; Lim, Jae-Min; Porterfield, Mindy; Liu, Mian; Satz, Jakob S; Buskirk, Sean; Xiong, Yufang; Zhang, Peng; Campbell, Kevin P; Hu, Huaiyu; Live, David; Tiemeyer, Michael; Wells, Lance

2011-06-17

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

A mild mutator phenotype arises in a mouse model for malignancies associated with neurofibromatosis type 1

International Nuclear Information System (INIS)

Garza, Rene; Hudson, Robert A.; McMahan, C. Alex; Walter, Christi A.; Vogel, Kristine S.

2007-01-01

Defects in genes that control DNA repair, proliferation, and apoptosis can increase genomic instability, and thus promote malignant progression. Although most tumors that arise in humans with neurofibromatosis type 1 (NF1) are benign, these individuals are at increased risk for malignant peripheral nerve sheath tumors (MPNST). To characterize additional mutations required for the development of MPNST from benign plexiform neurofibromas, we generated a mouse model for these tumors by combining targeted null mutations in Nf1 and p53, in cis. CisNf1+/-; p53+/- mice spontaneously develop PNST, and these tumors exhibit loss-of-heterozygosity at both the Nf1 and p53 loci. Because p53 has well-characterized roles in the DNA damage response, DNA repair, and apoptosis, and because DNA repair genes have been proposed to act as modifiers in NF1, we used the cisNf1+/-; p53+/- mice to determine whether a mutator phenotype arises in NF1-associated malignancies. To quantitate spontaneous mutant frequencies (MF), we crossed the Big Blue mouse, which harbors a lacI transgene, to the cisNf1+/-; p53+/- mice, and isolated genomic DNA from both tumor and normal tissues in compound heterozygotes and wild-type siblings. Many of the PNST exhibited increased mutant frequencies (MF = 4.70) when compared to normal peripheral nerve and brain (MF = 2.09); mutations occurred throughout the entire lacI gene, and included base substitutions, insertions, and deletions. Moreover, the brains, spleens, and livers of these cisNf1+/-; p53+/- animals exhibited increased mutant frequencies when compared to tissues from wild-type littermates. We conclude that a mild mutator phenotype arises in the tumors and tissues of cisNf1+/-; p53+/- mice, and propose that genomic instability influences NF1 tumor progression and disease severity

Enteric Neuron Imbalance and Proximal Dysmotility in Ganglionated Intestine of the Sox10Dom/+ Hirschsprung Mouse ModelSummary

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Melissa A. Musser

2015-01-01

Full Text Available Background & Aims: In Hirschsprung disease (HSCR, neural crest-derived progenitors (NCPs fail to completely colonize the intestine so that the enteric nervous system is absent from distal bowel. Despite removal of the aganglionic region, many HSCR patients suffer from residual intestinal dysmotility. To test the hypothesis that inappropriate lineage segregation of NCPs in proximal ganglionated regions of the bowel could contribute to such postoperative disease, we investigated neural crest (NC-derived lineages and motility in ganglionated, postnatal intestine of the Sox10Dom/+ HSCR mouse model. Methods: Cre-mediated fate-mapping was applied to evaluate relative proportions of NC-derived cell types. Motility assays were performed to assess gastric emptying and small intestine motility while colonic inflammation was assessed by histopathology for Sox10Dom/+ mutants relative to wild-type controls. Results: Sox10Dom/+ mice showed regional alterations in neuron and glia proportions as well as calretinin+ and neuronal nitric oxide synthase (nNOS+ neuronal subtypes. In the colon, imbalance of enteric NC derivatives correlated with the extent of aganglionosis. All Sox10Dom/+ mice exhibited reduced small intestinal transit at 4 weeks of age; at 6 weeks of age, Sox10Dom/+ males had increased gastric emptying rates. Sox10Dom/+ mice surviving to 6 weeks of age had little or no colonic inflammation when compared with wild-type littermates, suggesting that these changes in gastrointestinal motility are neurally mediated. Conclusions: The Sox10Dom mutation disrupts the balance of NC-derived lineages and affects gastrointestinal motility in the proximal, ganglionated intestine of adult animals. This is the first report identifying alterations in enteric neuronal classes in Sox10Dom/+ mutants, which suggests a previously unrecognized role for Sox10 in neuronal subtype specification. Keywords: Aganglionosis, Enteric Nervous System, Neural Crest

Conditional inactivation of TNFα-converting enzyme in chondrocytes results in an elongated growth plate and shorter long bones.

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Kenta Saito

Full Text Available TNFα-converting enzyme (TACE is a membrane-bound proteolytic enzyme with essential roles in the functional regulation of TNFα and epidermal growth factor receptor (EGFR ligands. Previous studies have demonstrated critical roles for TACE in vivo, including epidermal development, immune response, and pathological neoangiogenesis, among others. However, the potential contribution of TACE to skeletal development is still unclear. In the present study, we generated a Tace mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Histological analyses revealed that Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. Of note, we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice. Furthermore, we also found that phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13.

High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

Science.gov (United States)

Urban, Philippe; Truan, Gilles; Pompon, Denis

2014-01-01

The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Crystal structures of wild-type and mutated cyclophilin B that causes hyperelastosis cutis in the American quarter horse

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Boudko Sergei P

2012-11-01

Full Text Available Abstract Background Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results The crystal structures of both wild-type and mutated (Gly6->Arg horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions Interactions of the mutated cyclophilin B with a set of endoplasmic reticulum-resident proteins must be affected.

Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease

OpenAIRE

Toapanta, Franklin R.; Bernal, Paula J.; Fresnay, Stephanie; Darton, Thomas C.; Jones, Claire; Waddington, Claire S.; Blohmke, Christoph J.; Dougan, Gordon; Angus, Brian; Levine, Myron M.; Pollard, Andrew J.; Sztein, Marcelo B.

2015-01-01

A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD-) 5-10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12 h) and/or microbiological (S. Typhi bacteremia) endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e....

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Science.gov (United States)

Wong, Min Hao; Giraldo, Juan P; Kwak, Seon-Yeong; Koman, Volodymyr B; Sinclair, Rosalie; Lew, Tedrick Thomas Salim; Bisker, Gili; Liu, Pingwei; Strano, Michael S

2017-02-01

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm -1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Chronic activation of wild-type epidermal growth factor receptor and loss of Cdkn2a cause mouse glioblastoma formation.

Science.gov (United States)

Acquaviva, Jaime; Jun, Hyun Jung; Lessard, Julie; Ruiz, Rolando; Zhu, Haihao; Donovan, Melissa; Woolfenden, Steve; Boskovitz, Abraham; Raval, Ami; Bronson, Roderick T; Pfannl, Rolf; Whittaker, Charles A; Housman, David E; Charest, Al

2011-12-01

Glioblastoma multiforme (GBM) is characterized by overexpression of epidermal growth factor receptor (EGFR) and loss of the tumor suppressors Ink4a/Arf. Efforts at modeling GBM using wild-type EGFR in mice have proven unsuccessful. Here, we present a unique mouse model of wild-type EGFR-driven gliomagenesis. We used a combination of somatic conditional overexpression and ligand-mediated chronic activation of EGFR in cooperation with Ink4a/Arf loss in the central nervous system of adult mice to generate tumors with the histopathologic and molecular characteristics of human GBMs. Sustained, ligand-mediated activation of EGFR was necessary for gliomagenesis, functionally substantiating the clinical observation that EGFR-positive GBMs from patients express EGFR ligands. To gain a better understanding of the clinically disappointing EGFR-targeted therapies for GBM, we investigated the molecular responses to EGFR tyrosine kinase inhibitor (TKI) treatment in this model. Gefitinib treatment of primary GBM cells resulted in a robust apoptotic response, partially conveyed by mitogen-activated protein kinase (MAPK) signaling attenuation and accompanied by BIM(EL) expression. In human GBMs, loss-of-function mutations in the tumor suppressor PTEN are a common occurrence. Elimination of PTEN expression in GBM cells posttumor formation did not confer resistance to TKI treatment, showing that PTEN status in our model is not predictive. Together, these findings offer important mechanistic insights into the genetic determinants of EGFR gliomagenesis and sensitivity to TKIs and provide a robust discovery platform to better understand the molecular events that are associated with predictive markers of TKI therapy.

Impaired Latent Inhibition in GDNF-Deficient Mice Exposed to Chronic Stress

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Mona Buhusi

2017-10-01

Full Text Available Increased reactivity to stress is maladaptive and linked to abnormal behaviors and psychopathology. Chronic unpredictable stress (CUS alters catecholaminergic neurotransmission and remodels neuronal circuits involved in learning, attention and decision making. Glial-derived neurotrophic factor (GDNF is essential for the physiology and survival of dopaminergic neurons in substantia nigra and of noradrenergic neurons in the locus coeruleus. Up-regulation of GDNF expression during stress is linked to resilience; on the other hand, the inability to up-regulate GDNF in response to stress, as a result of either genetic or epigenetic modifications, induces behavioral alterations. For example, GDNF-deficient mice exposed to chronic stress exhibit alterations of executive function, such as increased temporal discounting. Here we investigated the effects of CUS on latent inhibition (LI, a measure of selective attention and learning, in GDNF-heterozygous (HET mice and their wild-type (WT littermate controls. No differences in LI were found between GDNF HET and WT mice under baseline experimental conditions. However, following CUS, GDNF-deficient mice failed to express LI. Moreover, stressed GDNF-HET mice, but not their WT controls, showed decreased neuronal activation (number of c-Fos positive neurons in the nucleus accumbens shell and increased activation in the nucleus accumbens core, both key regions in the expression of LI. Our results add LI to the list of behaviors affected by chronic stress and support a role for GDNF deficits in stress-induced pathological behaviors relevant to schizophrenia and other psychiatric disorders.

Hematopoietic Stem Cells from Ts65Dn Mice Are Deficient in the Repair of DNA Double-Strand Breaks.

Science.gov (United States)

Wang, Yingying; Chang, Jianhui; Shao, Lijian; Feng, Wei; Luo, Yi; Chow, Marie; Du, Wei; Meng, Aimin; Zhou, Daohong

2016-06-01

Down syndrome (DS) is a genetic disorder caused by the presence of an extra partial or whole copy of chromosome 21. In addition to musculoskeletal and neurodevelopmental abnormalities, children with DS exhibit various hematologic disorders and have an increased risk of developing acute lymphoblastic leukemia and acute megakaryocytic leukemia. Using the Ts65Dn mouse model, we investigated bone marrow defects caused by trisomy for 132 orthologs of the genes on human chromosome 21. The results showed that, although the total bone marrow cellularity as well as the frequency of hematopoietic progenitor cells (HPCs) was comparable between Ts65Dn mice and their age-matched euploid wild-type (WT) control littermates, human chromosome 21 trisomy led to a significant reduction in hematopoietic stem cell (HSC) numbers and clonogenic function in Ts65Dn mice. We also found that spontaneous DNA double-strand breaks (DSBs) were significantly increased in HSCs from the Ts65Dn mice, which was correlated with the significant reduction in HSC clonogenic activity compared to those from WT controls. Moreover, analysis of the repair kinetics of radiation-induced DSBs revealed that HSCs from Ts65Dn mice were less proficient in DSB repair than the cells from WT controls. This deficiency was associated with a higher sensitivity of Ts65Dn HSCs to radiation-induced suppression of HSC clonogenic activity than that of euploid HSCs. These findings suggest that an additional copy of genes on human chromosome 21 may selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies.

Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.

Science.gov (United States)

Burns, David P; Roy, Arijit; Lucking, Eric F; McDonald, Fiona B; Gray, Sam; Wilson, Richard J; Edge, Deirdre; O'Halloran, Ken D

2017-11-01

Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO 2 production is equivalent in wild-type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild-type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8-week-old male wild-type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force-generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO 2 production was not different between wild-type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild-type and

Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light

International Nuclear Information System (INIS)

Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

1998-01-01

The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition. (author)

Chinese herbal extract Su-duxing had potent inhibitory effects on both wild-type and entecavir-resistant hepatitis B virus (HBV) in vitro and effectively suppressed HBV replication in mouse model.

Science.gov (United States)

Liu, Yan; Yao, Weiming; Si, Lanlan; Hou, Jun; Wang, Jiabo; Xu, Zhihui; Li, Weijie; Chen, Jianhong; Li, Ruisheng; Li, Penggao; Bo, Lvping; Xiao, Xiaohe; Lan, Jinchu; Xu, Dongping

2018-04-24

The present study aimed to investigate anti-HBV effect and major active compounds of Su-duxing, a medicine extracted from Chinese herbs. HBV-replicating cell lines HepG2.2.15 (wild-type) and HepG2. A64 (entecavir-resistant) were used for in vitro test. C57BL/6 mice infected by adeno-associated virus carrying 1.3 mer wild-type HBV genome were used for in vivo test. Inhibitory rates of Su-duxing (10 μg/mL) on HBV replicative intermediate and HBsAg levels were 75.1%, 51.0% in HepG2.2.15 cells and 65.2%, 42.9% in HepG2. A64 cells. The 50% inhibitory concentration of Su-duxing and entecavir on HBV replicative intermediates had 0.2-fold and 712.5-fold increase respectively for entecavir-resistant HBV compared to wild-type HBV. Mice treated with Su-duxing (45.0 mg kg -1  d -1 for 2 weeks) had 1.39 log 10 IU/mL decrease of serum HBV DNA, and 48.9% and 51.7% decrease of serum HBsAg and HBeAg levels. GeneChip and KEGG analysis proposed that anti-HBV mechanisms included relief of HBx stability and viral replication, deregulation of early cell cycle checkpoints, and induction of type I interferon. Six active compounds (Matrine, Oxymatrine, Chlorogenic acid, Sophocarpine, Baicalein, and Wogonin) against HBV were identified in Su-duxing. Greater anti-HBV effects were observed in some compound pairs compared to each single compound. In conclusion, Su-duxing had potent inhibitory effects on both wild-type and entecavir-resistant HBV. Its effects were associated with coordinated roles of active compounds in its composition. Copyright © 2018. Published by Elsevier B.V.

Non-motor and motor features in LRRK2 transgenic mice.

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Zoë Bichler

Full Text Available Non-motor symptoms are increasingly recognized as important features of Parkinson's disease (PD. LRRK2 mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively evaluated in LRRK2 transgenic mouse models.Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor phenotypes precede motor symptoms.We investigated the onset of motor and non-motor phenotypes on the LRRK2(R1441G BAC transgenic mice and their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction, and learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate controls.LRRK2(R1441G BAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory mechanisms modulating the progression of PD in these models should be further evaluated.

Myg1-deficient mice display alterations in stress-induced responses and reduction of sex-dependent behavioural differences.

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Philips, Mari-Anne; Abramov, Urho; Lilleväli, Kersti; Luuk, Hendrik; Kurrikoff, Kaido; Raud, Sirli; Plaas, Mario; Innos, Jürgen; Puussaar, Triinu; Kõks, Sulev; Vasar, Eero

2010-02-11

Myg1 (Melanocyte proliferating gene 1) is a highly conserved and ubiquitously expressed gene, which encodes a protein with mitochondrial and nuclear localization. In the current study we demonstrate a gradual decline of Myg1 expression during the postnatal development of the mouse brain that suggests relevance for Myg1 in developmental processes. To study the effects of Myg1 loss-of-function, we created Myg1-deficient (-/-) mice by displacing the entire coding sequence of the gene. Initial phenotyping, covering a multitude of behavioural, cognitive, neurological, physiological and stress-related responses, revealed that homozygous Myg1 (-/-) mice are vital, fertile and display no gross abnormalities. Myg1 (-/-) mice showed an inconsistent pattern of altered anxiety-like behaviour in different tests. The plus-maze and social interaction tests revealed that male Myg1 (-/-) mice were significantly less anxious than their wild-type littermates; female (-/-) mice showed increased anxiety in the locomotor activity arena. Restraint-stress significantly reduced the expression of the Myg1 gene in the prefrontal cortex of female wild-type mice and restrained female (-/-) mice showed a blunted corticosterone response, suggesting involvement of Myg1 in stress-induced responses. The main finding of the present study was that Myg1 invalidation decreases several behavioural differences between male and female animals that were obvious in wild-type mice, indicating that Myg1 contributes to the expression of sex-dependent behavioural differences in mice. Taken together, we provide evidence for the involvement of Myg1 in anxiety- and stress-related responses and suggest that Myg1 contributes to the expression of sex-dependent behavioural differences.

Ontogenic studies of the neural control of adenohypophyseal hormones in the rat: gonadotropins.

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Becú-Villalobos, D; Lacau-Mengido, I M; Libertun, C

1990-12-01

1. Serotonergic, dopaminergic, and opioid systems controlling luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion develop with particular characteristics in the male and female prepubertal rats. 2. Serotonergic pathways evoke a maximal release of LH and FSH in female rats from day 12 to day 20 of age, but not in males of the same age. 3. Antidopaminergic drugs increase LH and FSH levels only in the female infantile rats. This effect is absent at birth and disappears after 20 days of age. 4. Naloxone markedly increases gonadotropins in 12-day-old females. 5. On the other hand, in 12-day-old male rats some neurotropic drugs such as diazepam could enhance LH levels, the effect being absent at other ages or in female littermates. 6. A period of high sensitivity of gonadotropins to neurotropic drugs is present during the second and third weeks of life of the rat and it is related to the sexual differentiation of the brain.

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells

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van Benthem Jan

2010-01-01

Full Text Available Abstract Background Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC as compared to wild-type (WT cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM and γ-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU and vehicle were taken as controls. Results Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments. Conclusion These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

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Philippe Urban

2014-01-01

Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

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Ilany, Jacob; Bilan, Philip J.; Kapur, Sonia; Caldwell, James S.; Patti, Mary-Elizabeth; Marette, Andre; Kahn, C. Ronald

2006-03-01

Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes. diabetes mellitus | glucose transport | RGK GTPase | transgenic mouse

Aberrant Bone Density in Aging Mice Lacking the Adenosine Transporter ENT1

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Hinton, David J.; McGee-Lawrence, Meghan E.; Lee, Moonnoh R.; Kwong, Hoi K.; Westendorf, Jennifer J.; Choi, Doo-Sup

2014-01-01

Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1) is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months) compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density. PMID:24586402

Aberrant bone density in aging mice lacking the adenosine transporter ENT1.

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David J Hinton

Full Text Available Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1 is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP, an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density.

PKCepsilon overexpression, irrespective of genetic background, sensitizes skin to UVR-induced development of squamous-cell carcinomas.

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Sand, Jordan M; Aziz, Moammir H; Dreckschmidt, Nancy E; Havighurst, Thomas C; Kim, KyungMann; Oberley, Terry D; Verma, Ajit K

2010-01-01

Chronic exposure to UVR is the major etiologic factor in the development of human skin cancers including squamous-cell carcinoma (SCC). We have previously shown that protein Kinase C epsilon (PKCepsilon) transgenic mice on FVB/N background, which overexpress PKCepsilon protein approximately eightfold over endogenous levels in epidermis, exhibit about threefold more sensitivity than wild-type littermates to UVR-induced development of SCC. To determine whether it is PKCepsilon and not the mouse genetic background that determines susceptibility to UVR carcinogenesis, we cross-bred PKCepsilon FVB/N transgenic mice with SKH-1 hairless mice to generate PKCepsilon-overexpressing SKH-1 hairless mice. To evaluate the susceptibility of PKCepsilon SKH-1 hairless transgenic mice to UVR carcinogenesis, the mice were exposed to UVR (1-2 KJ m(-2)) three times weekly from a bank of six kodacel-filtered FS40 sunlamps. As compared with the wild-type hairless mice, PKCepsilon overexpression in SKH-1 hairless mice decreased the latency (12 weeks), whereas it increased the incidence (twofold) and multiplicity (fourfold) of SCC. The SKH hairless transgenic mice were observed to be as sensitive as FVB/N transgenic mice to UVR-induced development of SCC and expression of proliferative markers (proliferating cell nuclear antigen, signal transducers and activators of transcription 3, and extracellular signal-regulated kinase 1/2). The results indicate that PKCepsilon level dictates susceptibility, irrespective of genetic background, to UVR carcinogenesis.

Plasma adiponectin levels are increased despite insulin resistance in corticotropin-releasing hormone transgenic mice, an animal model of Cushing syndrome.

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Shinahara, Masayuki; Nishiyama, Mitsuru; Iwasaki, Yasumasa; Nakayama, Shuichi; Noguchi, Toru; Kambayashi, Machiko; Okada, Yasushi; Tsuda, Masayuki; Stenzel-Poore, Mary P; Hashimoto, Kozo; Terada, Yoshio

2009-01-01

Adiponectin (AdN), an adipokine derived from the adipose tissue, has an insulin-sensitizing effect, and plasma AdN is shown to be decreased in obesity and/or insulin resistant state. To clarify whether changes in AdN are also responsible for the development of glucocorticoid-induced insulin resistance, we examined AdN concentration in plasma and AdN expression in the adipose tissue, using corticotropin-releasing hormone (CRH) transgenic mouse (CRH-Tg), an animal model of Cushing syndrome. We found, unexpectedly, that plasma AdN levels in CRHTg were significantly higher than those in wild-type littermates (wild-type: 19.7+/-2.5, CRH-Tg: 32.4+/-3.1 microg/mL, pAdN mRNA and protein levels were significantly decreased in the adipose tissue of CRH-Tg. Bilateral adrenalectomy in CRH-Tg eliminated both their Cushing's phenotype and their increase in plasma AdN levels (wild-type/sham: 9.4+/-0.5, CRH-Tg/sham: 15.7+/-2.0, CRH-Tg/ADX: 8.5+/-0.4 microg/mL). These results strongly suggest that AdN is not a major factor responsible for the development of insulin resistance in Cushing syndrome. Our data also suggest that glucocorticoid increases plasma AdN levels but decreases AdN expression in adipocytes, the latter being explained possibly by the decrease in AdN metabolism in the Cushing state.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice.

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McMahon, C D; Chai, R; Radley-Crabb, H G; Watson, T; Matthews, K G; Sheard, P W; Soffe, Z; Grounds, M D; Shavlakadze, T

2014-12-01

The age-related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin-like growth factor-1 (IGF-1). We hypothesized that constitutively expressed IGF-1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF-1 Ea in skeletal muscles were compared with wild-type littermates. Four-month-old mice were assigned to be sedentary, or had access to free-running wheels, until 18 or 28 months of age. In wild-type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF-1 alone was ineffective, whereas the combination of exercise and IGF-1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF-1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF-1 have a modest effect on reducing aged-related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Differentially regulated protein kinase A (PKA) activity in adipose tissue and liver is associated with resistance to diet-induced obesity and glucose intolerance in mice that lack PKA regulatory subunit type IIα.

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London, Edra; Nesterova, Maria; Sinaii, Ninet; Szarek, Eva; Chanturiya, Tatyana; Mastroyannis, Spyridon A; Gavrilova, Oksana; Stratakis, Constantine A

2014-09-01

The cAMP-dependent protein kinase A (PKA) signaling system is widely expressed and has a central role in regulating cellular metabolism in all organ systems affected by obesity. PKA has four regulatory (RIα, RIIα, RIβ, RIIβ) and four catalytic (Cα, Cβ, Cγ, Prkx) subunit isoforms that have tissue-specific expression profiles. In mice, knockout (KO) of RIIβ, the primary PKA regulatory subunit in adipose tissue or knockout of the catalytic subunit Cβ resulted in a lean phenotype that resists diet-induced obesity and associated metabolic complications. Here we report that the disruption of the ubiquitously expressed PKA RIIα subunit in mice (RIIαKO) confers resistance to diet-induced obesity, glucose intolerance, and hepatic steatosis. After 2-week high-fat diet exposure, RIIαKO mice weighed less than wild-type littermates. Over time this effect was more pronounced in female mice that were also leaner than their wild-type counterparts, regardless of the diet. Decreased intake of a high-fat diet contributed to the attenuated weight gain in RIIαKO mice. Additionally, RIIα deficiency caused differential regulation of PKA in key metabolic organs: cAMP-stimulated PKA activity was decreased in liver and increased in gonadal adipose tissue. We conclude that RIIα represents a potential target for therapeutic interventions in obesity, glucose intolerance, and nonalcoholic fatty liver disease.

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

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David J Speca

2010-08-01

Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Increased Hepatic Expression of Endothelial Lipase Inhibits Cholesterol Diet-Induced Hypercholesterolemia and Atherosclerosis in Transgenic Rabbits.

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Wang, Chuan; Nishijima, Kazutoshi; Kitajima, Shuji; Niimi, Manabu; Yan, Haizhao; Chen, Yajie; Ning, Bo; Matsuhisa, Fumikazu; Liu, Enqi; Zhang, Jifeng; Chen, Y Eugene; Fan, Jianglin

2017-07-01

Endothelial lipase (EL) is a key determinant in plasma high-density lipoprotein-cholesterol. However, functional roles of EL on the development of atherosclerosis have not been clarified. We investigated whether hepatic expression of EL affects plasma lipoprotein metabolism and cholesterol diet-induced atherosclerosis. We generated transgenic (Tg) rabbits expressing the human EL gene in the liver and then examined the effects of EL expression on plasma lipids and lipoproteins and compared the susceptibility of Tg rabbits with cholesterol diet-induced atherosclerosis with non-Tg littermates. On a chow diet, hepatic expression of human EL in Tg rabbits led to remarkable reductions in plasma levels of total cholesterol, phospholipids, and high-density lipoprotein-cholesterol compared with non-Tg controls. On a cholesterol-rich diet for 16 weeks, Tg rabbits exhibited significantly lower hypercholesterolemia and less atherosclerosis than non-Tg littermates. In Tg rabbits, gross lesion area of aortic atherosclerosis was reduced by 52%, and the lesions were characterized by fewer macrophages and smooth muscle cells compared with non-Tg littermates. Increased hepatic expression of EL attenuates cholesterol diet-induced hypercholesterolemia and protects against atherosclerosis. © 2017 American Heart Association, Inc.

Expressions of tight junction proteins Occludin and Claudin-1 are under the circadian control in the mouse large intestine: implications in intestinal permeability and susceptibility to colitis.

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Oh-oka Kyoko

Full Text Available BACKGROUND & AIMS: The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine. METHODS: The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2(m/m. In addition, the susceptibility to dextran sodium sulfate (DSS-induced colitis was compared between wild-type mice and mPer2(m/m mice. RESULTS: The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2(m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2(m/m mice. mPer2(m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice. CONCLUSIONS: Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.

The p27 Pathway Modulates the Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone-Related Peptide.

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Zhu, Min; Zhang, Jing; Dong, Zhan; Zhang, Ying; Wang, Rong; Karaplis, Andrew; Goltzman, David; Miao, Dengshun

2015-11-01

Parathyroid hormone-related peptide (PTHrP) 1-84 knock-in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild-type (WT) littermates. We found that the expression levels of p27, p16, and p53 were significantly upregulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice, which were homozygous for both p27 deletion and the Pthrp KI mutation (p27(-/-) Pthrp KI). We then compared p27(-/-) Pthrp KI mice with p27(-/-), Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight, and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of proliferating cell nuclear antigen (PCNA)-positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and alkaline phosphatase (ALP)-, type I collagen-, and osteocalcin-positive bone areas were increased in p27(-/-) mice and reduced in both Pthrp KI and p27(-/-) Pthrp KI mice compared with WT mice; however, these parameters were increased in p27(-/-) Pthrp KI mice compared with Pthrp KI mice. As well, protein expression levels of PTHR, IGF-1, and Bmi-1, and the numbers of total colony-forming unit fibroblastic (CFU-f) and ALP-positive CFU-f were similarly increased in p27(-/-) Pthrp KI mice compared with Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies, therefore, indicate that the p27 pathway may function downstream in the action

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain

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Masoud Keighobadi

2018-03-01

Full Text Available Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51, which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes (p-value < 0.05 in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were −40,027.1 and −39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

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Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Control of blood pressure, appetite, and glucose by leptin in mice lacking leptin receptors in proopiomelanocortin neurons.

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do Carmo, Jussara M; da Silva, Alexandre A; Cai, Zhengwei; Lin, Shuying; Dubinion, John H; Hall, John E

2011-05-01

Although the central nervous system melanocortin system is an important regulator of energy balance, the role of proopiomelanocortin (POMC) neurons in mediating the chronic effects of leptin on appetite, blood pressure, and glucose regulation is unknown. Using Cre/loxP technology we tested whether leptin receptor deletion in POMC neurons (LepR(flox/flox)/POMC-Cre mice) attenuates the chronic effects of leptin to increase mean arterial pressure (MAP), enhance glucose use and oxygen consumption, and reduce appetite. LepR(flox/flox)/POMC-Cre, wild-type, LepR(flox/flox), and POMC-Cre mice were instrumented for MAP and heart rate measurement by telemetry and venous catheters for infusions. LepR(flox/flox)/POMC-Cre mice were heavier, hyperglycemic, hyperinsulinemic, and hyperleptinemic compared with wild-type, LepR(flox/flox), and POMC-Cre mice. Despite exhibiting features of metabolic syndrome, LepR(flox/flox)/POMC-Cre mice had normal MAP and heart rate compared with LepR(flox/flox) but lower MAP and heart rate compared with wild-type mice. After a 5-day control period, leptin was infused (2 μg/kg per minute, IV) for 7 days. In control mice, leptin increased MAP by ≈5 mm Hg despite decreasing food intake by ≈35%. In contrast, leptin infusion in LepR(flox/flox)/POMC-Cre mice reduced MAP by ≈3 mm Hg and food intake by ≈28%. Leptin significantly decreased insulin and glucose levels in control mice but not in LepR(flox/flox)/POMC-Cre mice. Leptin increased oxygen consumption in LepR(flox/flox)/POMC-Cre and wild-type mice. Activation of POMC neurons is necessary for the chronic effects of leptin to raise MAP and reduce insulin and glucose levels, whereas leptin receptors in other areas of the brain other than POMC neurons appear to play a key role in mediating the chronic effects of leptin on appetite and oxygen consumption.

Portal control of viral prohead expansion and DNA packaging

International Nuclear Information System (INIS)

Ray, Krishanu; Oram, Mark; Ma, Jinxia; Black, Lindsay W.

2009-01-01

Bacteriophage T4 terminase packages DNA in vitro into empty small or large proheads (esps or elps). In vivo maturation of esps yields the more stable and voluminous elps required to contain the 170 kb T4 genome. Functional proheads can be assembled containing portal-GFP fusion proteins. In the absence of terminase activity these accumulated in esps in vivo, whereas wild-type portals were found in elps. By nuclease protection assay dsDNAs of lengths 0.1, 0.2, 0.5, 5, 11, 20, 40 or 170 kb were efficiently packaged into wild-type elps in vitro, but less so into esps and gp20-GFP elps; particularly with DNAs shorter than 11 kb. However, 0.1 kb substrates were equally efficiently packaged into all types of proheads as judged by fluorescence correlation spectroscopy. These data suggest the portal controls the expansion of the major capsid protein lattice during prohead maturation, and that this expansion is necessary for DNA protection but not for packaging.

Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states

Energy Technology Data Exchange (ETDEWEB)

Bodenheimer, Annette M. [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA; Meilleur, Flora [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA

2016-11-07

Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice

NARCIS (Netherlands)

Sinha, Pratima; Chi, Howard H.; Kim, Hong R.; Clausen, Björn E.; Pederson, Brian; Sercarz, Eli E.; Forster, Irmgard; Moudgil, Kamal D.

2004-01-01

We have studied T cell tolerance to defined determinants within ML-M using wild-type (WT; ML-M+/+) and LysMcre (ML-M-/-) C3H (H-2(k)) mice to determine the relative contribution of ML-M-derived epitopes vs those from other self Ags in selection of the ML-M-specific T cell repertoire. ML-M was

Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1,000 Genomes Project.

Science.gov (United States)

Santpere, Gabriel; Darre, Fleur; Blanco, Soledad; Alcami, Antonio; Villoslada, Pablo; Mar Albà, M; Navarro, Arcadi

2014-04-01

Most people in the world (∼90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis, and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomic analysis of EBV including, for the first time, EBV strains derived from healthy individuals, we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. As strain B95-8 was used to transform B cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605), we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbor more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging, and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite coevolution.

Multicenter Phase II study of FOLFOX or biweekly XELOX and Erbitux (cetuximab) as first-line therapy in patients with wild-type KRAS/BRAF metastatic colorectal cancer: The FLEET study

International Nuclear Information System (INIS)

Soda, Hitoshi; Maeda, Hiromichi; Hasegawa, Junichi; Takahashi, Takao; Hazama, Shoichi; Fukunaga, Mutsumi; Kono, Emiko; Kotaka, Masahito; Sakamoto, Junichi; Nagata, Naoki; Oba, Koji; Mishima, Hideyuki

2015-01-01

The clinical benefit of cetuximab combined with oxaliplatin-based chemotherapy remains under debate. The aim of the present multicenter open-label Phase II study was to explore the efficacy and safety of biweekly administration of cetuximab and mFOLFOX-6 or XELOX as first-line chemotherapy in patients with metastatic colorectal cancer. Sixty-two patients with previously untreated KRAS/BRAF wild-type metastatic colorectal cancer were recruited to the study between April 2010 and May 2011. Patients received one of two treatment regimens, either cetuximab plus mFOLFOX-6 (FOLFOX + Cmab) or cetuximab plus biweekly XELOX (XELOX + Cmab), according to their own preference. Treatment was continued until disease progression or the appearance of intolerable toxicities. The primary endpoint was response rate; secondary endpoints were progression-free survival, overall survival, disease control rate, dose intensity, conversion rate to surgical resection, and safety. The response rates in the FOLFOX + Cmab (n = 37) and XELOX + Cmab (n = 25) groups were 64.9 % (24/37) and 72.0 % (18/25), respectively. The median PFS in the FOLFOX + Cmab and XELOX + Cmab groups was 13.1 months (95 % confidence interval [CI] 12.1–17.5) and 13.4 months (95 % CI 10.1–17.9), respectively. Neutropenia was the most frequent grade 3/4 adverse event in both groups (33.9 %), followed by anorexia, acneiform eruption, skin fissure and paronychia. A waterfall plot of tumor diameter showed prominent shrinkage of the tumors in 88.7 % of patients. The results of the present study indicate that biweekly cetuximab plus mFOLFOX-6/XELOX is an effective and tolerable treatment regimen. Biweekly administration of cetuximab requires only one hospital visit every 2 weeks, and may become a convenient treatment option for patients with KRAS/BRAF wild-type metastatic colorectal cancer

No Overt Deficits in Aged Tau-Deficient C57Bl/6.Mapttm1(EGFPKit GFP Knockin Mice.

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Annika van Hummel

Full Text Available Several mouse lines with knockout of the tau-encoding MAPT gene have been reported in the past; they received recent attention due to reports that tau reduction prevented Aβ-induced deficits in mouse models of Alzheimer's disease. However, the effects of long-term depletion of tau in vivo remained controversial. Here, we used the tau-deficient GFP knockin line Mapttm1(EGFPkit on a pure C57Bl/6 background and subjected a large cohort of males and females to a range of motor, memory and behavior tests and imaging analysis, at the advanced age of over 16 months. Neither heterozygous nor homozygous Mapttm1(EGFPkit mice presented with deficits or abnormalities compared to wild-type littermates. Differences to reports using other tau knockout models may be due to different genetic backgrounds, respective gene targeting strategies or other confounding factors, such as nutrition. To this end, we report no functional or morphological deficits upon tau reduction or depletion in aged mice.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Science.gov (United States)

Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

2014-03-04

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Reduction of age-associated pathology in old mice by overexpression of catalase in mitochondria.

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Treuting, Piper M; Linford, Nancy J; Knoblaugh, Sue E; Emond, M J; Morton, John F; Martin, George M; Rabinovitch, Peter S; Ladiges, Warren C

2008-08-01

We describe the effects of mitochondrially targeted catalase (MCAT) expression on end-of-life pathology in mice using detailed semiquantitative histopathological evaluation. We previously reported that the median and maximum life spans of MCAT mice were extended relative to those of wild-type littermates. We now report that MCAT expression is associated with reduced malignant nonhematopoietic tumor burden, reduced cardiac lesions, and a trend toward reduced systemic inflammation, with no effect on hematopoietic neoplasia or glomerulonephropathy. Combined disease burden and comorbidity are also reduced, and MCAT expression is not associated with any detrimental clinical effects. The results suggest that oxidative damage is involved in aging of C57BL/6J mice via modulation of a subset of age-associated lesions. Antioxidant interventions targeting mitochondria may therefore be a viable strategy for prevention or postponement of some age-associated diseases. The variability of the MCAT effect across tissues, however, illustrates the importance of developing semiquantitative histopathology for assessment of comorbidity in life-span studies.

Transgenic overexpression of p23 induces spontaneous hydronephrosis in mice

Science.gov (United States)

Lee, Jaehoon; Kim, Hye Jin; Moon, Jung Ah; Sung, Young Hoon; Baek, In-Jeoung; Roh, Jae-il; Ha, Na Young; Kim, Seung-Yeon; Bahk, Young Yil; Lee, Jong Eun; Yoo, Tae Hyun; Lee, Han-Woong

2011-01-01

p23 is a cochaperone of heat shock protein 90 and also interacts functionally with numerous steroid receptors and kinases. However, the in vivo roles of p23 remain unclear. To explore its in vivo function, we generated the transgenic (TG) mice ubiquitously overexpressing p23. The p23 TG mice spontaneously developed kidney abnormalities closely resembling human hydronephrosis. Consistently, kidney functions deteriorate significantly in the p23 TG mice compared to their wild-type (WT) littermates. Furthermore, the expression of target genes for aryl hydrocarbon receptor (AhR), such as cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1A1) and cytochrome P450, family 1, subfamily B, polypeptide 1 (Cyp1B1), were induced in the kidneys of the p23 TG mice. These results indicate that the overexpression of p23 contributes to the development of hydronephrosis through the upregulation of the AhR pathway in vivo. PMID:21323770

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

DEFF Research Database (Denmark)

Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

2009-01-01

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

PGC-1α and exercise intensity dependent adaptations in mouse skeletal muscle

DEFF Research Database (Denmark)

Brandt, Nina; Dethlefsen, Maja Munk; Bangsbo, Jens

2017-01-01

The aim of the present study was to examine the role of PGC-1α in intensity dependent exercise and exercise training-induced metabolic adaptations in mouse skeletal muscle. Whole body PGC-1α knockout (KO) and littermate wildtype (WT) mice performed a single treadmill running bout at either low...... intensity dependent increases in LC3I and LC3II protein and intensity independent decrease in p62 protein in skeletal muscle late in recovery and increased LC3II with exercise training independent of exercise intensity and volume in WT mice. Furthermore, acute exercise and exercise training did not increase...... LC3I and LC3II protein in PGC-1α KO. In addition, exercise-induced mRNA responses of PGC-1α isoforms were intensity dependent. In conclusion, these findings indicate that exercise intensity affected autophagy markers differently in skeletal muscle and suggest that PGC-1α regulates both acute...

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

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Ashlee J. Conway

2017-08-01

Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

Science.gov (United States)

Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

2012-01-01

The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

NARCIS (Netherlands)

Dummer, R; Bergh, J; Karlsson, Y; Horovitz, JA; Mulder, NH; Huinin, DT; Burg, G; Hofbauer, G; Osanto, S

p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector

Elevated Incidence of Dental Caries in a Mouse Model of Cystic Fibrosis

Science.gov (United States)

Catalán, Marcelo A.; Scott-Anne, Kathleen; Klein, Marlise I.; Koo, Hyun; Bowen, William H.; Melvin, James E.

2011-01-01

Background Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model. Methodology/Principal Findings We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value<0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16±2 mM vs. 31±2 mM, CF and wildtype mice, respectively; p value<0.01; t test). Conclusions/Significance Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse. PMID:21304986

Elevated incidence of dental caries in a mouse model of cystic fibrosis.

Directory of Open Access Journals (Sweden)

Marcelo A Catalán

2011-01-01

Full Text Available Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF. Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model.We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000. The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value < 0.003; t test. We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16 ± 2 mM vs. 31 ± 2 mM, CF and wildtype mice, respectively; p value < 0.01; t test.Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.

Genetic management strategies for controlling infectious diseases in livestock populations

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Bishop Stephen C

2003-06-01

Full Text Available Abstract This paper considers the use of disease resistance genes to control the transmission of infection through an animal population. Transmission is summarised by R0, the basic reproductive ratio of a pathogen. If R0 > 1.0 a major epidemic can occur, thus a disease control strategy should aim to reduce R0 below 1.0, e.g. by mixing resistant with susceptible wild-type animals. Suppose there is a resistance allele, such that transmission of infection through a population homozygous for this allele will be R02 01, where R01 describes transmission in the wildtype population. For an otherwise homogeneous population comprising animals of these two groups, R0 is the weighted average of the two sub-populations: R0 = R01ρ + R02 (1 - ρ, where ρ is the proportion of wildtype animals. If R01 > 1 and R02 0 ≤ 1, i.e. ρ ≤ (R0 - R02/(R01 - R02. If R02 = 0, the proportion of resistant animals must be at least 1 - 1/R01. For an n genotype model the requirement is still to have R0 ≤ 1.0. Probabilities of epidemics in genetically mixed populations conditional upon the presence of a single infected animal were derived. The probability of no epidemic is always 1/(R0 + 1. When R0 ≤ 1 the probability of a minor epidemic, which dies out without intervention, is R0/(R0 + 1. When R0 > 1 the probability of a minor and major epidemics are 1/(R0 + 1 and (R0 - 1/(R0 + 1. Wherever possible a combination of genotypes should be used to minimise the invasion possibilities of pathogens that have mutated to overcome the effects of specific resistance alleles.

Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation.

Science.gov (United States)

Desai, Harsh R; Sivasubramaniyam, Tharini; Revelo, Xavier S; Schroer, Stephanie A; Luk, Cynthia T; Rikkala, Prashanth R; Metherel, Adam H; Dodington, David W; Park, Yoo Jin; Kim, Min Jeong; Rapps, Joshua A; Besla, Rickvinder; Robbins, Clinton S; Wagner, Kay-Uwe; Bazinet, Richard P; Winer, Daniel A; Woo, Minna

2017-08-09

During obesity, macrophages can infiltrate metabolic tissues, and contribute to chronic low-grade inflammation, and mediate insulin resistance and diabetes. Recent studies have elucidated the metabolic role of JAK2, a key mediator downstream of various cytokines and growth factors. Our study addresses the essential role of macrophage JAK2 in the pathogenesis to obesity-associated inflammation and insulin resistance. During high-fat diet (HFD) feeding, macrophage-specific JAK2 knockout (M-JAK2 -/- ) mice gained less body weight compared to wildtype littermate control (M-JAK2 +/+ ) mice and were protected from HFD-induced systemic insulin resistance. Histological analysis revealed smaller adipocytes and qPCR analysis showed upregulated expression of some adipogenesis markers in visceral adipose tissue (VAT) of HFD-fed M-JAK2 -/- mice. There were decreased crown-like structures in VAT along with reduced mRNA expression of some macrophage markers and chemokines in liver and VAT of HFD-fed M-JAK2 -/- mice. Peritoneal macrophages from M-JAK2 -/- mice and Jak2 knockdown in macrophage cell line RAW 264.7 also showed lower levels of chemokine expression and reduced phosphorylated STAT3. However, leptin-dependent effects on augmenting chemokine expression in RAW 264.7 cells did not require JAK2. Collectively, our findings show that macrophage JAK2 deficiency improves systemic insulin sensitivity and reduces inflammation in VAT and liver in response to metabolic stress.

Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

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Niurka Trujillo-Paredes

2016-03-01

Full Text Available Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs, but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+. These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons.

Fetal brain 11β-hydroxysteroid dehydrogenase type 2 selectively determines programming of adult depressive-like behaviors and cognitive function, but not anxiety behaviors in male mice.

Science.gov (United States)

Wyrwoll, Caitlin; Keith, Marianne; Noble, June; Stevenson, Paula L; Bombail, Vincent; Crombie, Sandra; Evans, Louise C; Bailey, Matthew A; Wood, Emma; Seckl, Jonathan R; Holmes, Megan C

2015-09-01

Stress or elevated glucocorticoids during sensitive windows of fetal development increase the risk of neuropsychiatric disorders in adult rodents and humans, a phenomenon known as glucocorticoid programming. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyses rapid inactivation of glucocorticoids in the placenta, controls access of maternal glucocorticoids to the fetal compartment, placing it in a key position to modulate glucocorticoid programming of behavior. However, the importance of the high expression of 11β-HSD2 within the midgestational fetal brain is unknown. To examine this, a brain-specific knockout of 11β-HSD2 (HSD2BKO) was generated and compared to wild-type littermates. HSD2BKO have markedly diminished fetal brain 11β-HSD2, but intact fetal body and placental 11β-HSD2 and normal fetal and placental growth. Despite normal fetal plasma corticosterone, HSD2BKO exhibit elevated fetal brain corticosterone levels at midgestation. As adults, HSD2BKO show depressive-like behavior and have cognitive impairments. However, unlike complete feto-placental deficiency, HSD2BKO show no anxiety-like behavioral deficits. The clear mechanistic separation of the programmed components of depression and cognition from anxiety implies distinct mechanisms of pathogenesis, affording potential opportunities for stratified interventions. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

Science.gov (United States)

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

Science.gov (United States)

Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.

Adiponectin protects against development of metabolic disturbances in a PCOS mouse model.

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Benrick, Anna; Chanclón, Belén; Micallef, Peter; Wu, Yanling; Hadi, Laila; Shelton, John M; Stener-Victorin, Elisabet; Wernstedt Asterholm, Ingrid

2017-08-22

Adiponectin, together with adipocyte size, is the strongest factor associated with insulin resistance in women with polycystic ovary syndrome (PCOS). This study investigates the causal relationship between adiponectin levels and metabolic and reproductive functions in PCOS. Prepubertal mice overexpressing adiponectin from adipose tissue (APNtg), adiponectin knockouts (APNko), and their wild-type (WT) littermate mice were continuously exposed to placebo or dihydrotestosterone (DHT) to induce PCOS-like traits. As expected, DHT exposure led to reproductive dysfunction, as judged by continuous anestrus, smaller ovaries with a decreased number of corpus luteum, and an increased number of cystic/atretic follicles. A two-way between-groups analysis showed that there was a significant main effect for DHT exposure, but not for genotype, indicating adiponectin does not influence follicle development. Adiponectin had, however, some protective effects on ovarian function. Similar to in many women with PCOS, DHT exposure led to reduced adiponectin levels, larger adipocyte size, and reduced insulin sensitivity in WTs. APNtg mice remained metabolically healthy despite DHT exposure, while APNko-DHT mice were even more insulin resistant than their DHT-exposed littermate WTs. DHT exposure also reduced the mRNA expression of genes involved in metabolic pathways in gonadal adipose tissue of WT and APNko, but this effect of DHT was not observed in APNtg mice. Moreover, APNtg-DHT mice displayed increased pancreatic mRNA levels of insulin receptors, Pdx1 and Igf1R , suggesting adiponectin stimulates beta cell viability/hyperplasia in the context of PCOS. In conclusion, adiponectin improves metabolic health but has only minor effects on reproductive functions in this PCOS-like mouse model.

Characterization and comparison of transgenic Artemisia annua GYR and wild-type NON-GYR plants in an environmental release trial.

Science.gov (United States)

Liu, H; Wu, G G; Wang, J B; Wu, X; Bai, L; Jiang, W; Lv, B B; Pan, A H; Jia, J W; Li, P; Zhao, K; Jiang, L X; Tang, X M

2016-08-26

The anti-malarial drug, artemisinin, is quite expensive as a result of its slow content in Artemisia annua. Recent investigations have suggested that genetic engineering of A. annua is a promising approach to improve the yield of artemisinin. In this study, the transgenic A. annua strain GYR, which has high artemisinin content, was evaluated in an environmental release trial. First, GYR plants were compared with the wild-type variety NON-GYR, with regard to phenotypic characters (plant height, crown width, stem diameter, germination rate, leaf dry weight, 1000-seed weight, leave shape). Second, stress resistance in the two varieties (salt, drought, herbicide, and cold resistance) was evaluated under different experimental conditions. Finally, gene flow was estimated. The results indicated that there were significant differences in several agronomic traits (plant height, stem diameter, and leave dry weight) between the transgenic GYR and NON-GYR plants. Salt stress in transgenic and control plants was similar, except under high NaCl concentrations (1.6%, w/w). Leaf water, proline, and MDA content (increased significantly) were significantly different. Transgenic A. annua GYR plants did not grow better than NON-GYR plants with respect to drought and herbicide resistance. The two varieties maintained vitality through the winter. Third, gene flow was studied in an environmental risk trial for transgenic GYR. The maximum gene flow frequency was 2.5%, while the maximum gene flow distance was 24.4 m; gene flow was not detected at 29.2 m at any direction. Our findings may provide an opportunity for risk assessment in future commercialization of transgenic A. annua varieties.

The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells : comparison of immunocytochemical determination with detection in isolated DNA

NARCIS (Netherlands)

Blommaert, F.A.; Floot, B.G.J.; Dijk-Knijnenburg, H.C.M. van; Berends, F.; Baan, R.A.; Schornagel, J.H.; Engelse, L. den; Fichtinger-Schepman, A.M.J.

1998-01-01

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively.

Synovial deposition of wild-type transthyretin-derived amyloid in knee joint osteoarthritis patients.

Science.gov (United States)

Takanashi, Tetsuo; Matsuda, Masayuki; Yazaki, Masahide; Yamazaki, Hideshi; Nawata, Masashi; Katagiri, Yoshiki; Ikeda, Shu-Ichi

2013-09-01

To investigate histological features of deposited amyloid in the synovial tissue and its clinical significance in knee joint osteoarthritis (OA) patients. We prospectively enrolled 232 consecutive patients who underwent arthroplasty or total replacement of the knee joint for treatment of OA. Congo red staining and immunohistochemistry were performed in the synovial tissue obtained at surgery. When transthyretin (TTR)-derived amyloid was positive, we analyzed all 4 exons of the TTR gene using the direct DNA sequencing method in order to detect mutations. We analyzed 322 specimens in this study. Twenty-six specimens (8.1%) obtained from 21 patients (5 men and 16 women; mean, 79.0 ± 4.6 years) showed deposition of amyloid, which was positively stained with the anti-TTR antibody. Eighteen patients showed inhomogeneous accumulations of amyloid in the loose connective tissue under the synovial epithelia sometimes with nodule formation, while in the remaining three, small vessels in the adipose tissue were involved. Medical records of these patients revealed nothing remarkable in the clinical course, laboratory data or macroscopic intraarticular findings at surgery. No mutations were detectable in the TTR gene analysis. Wild-type TTR-derived amyloid may affect the synovial tissue as a result of long-term mechanical stress or as a part of senile systemic amyloidosis in approximately 8% of knee joint OA patients. No obvious clinical significance was found in synovial deposition of amyloid.

Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.

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Xu, Bing; Jiang, Mingzuo; Chu, Yi; Wang, Weijie; Chen, Di; Li, Xiaowei; Zhang, Zhao; Zhang, Di; Fan, Daiming; Nie, Yongzhan; Shao, Feng; Wu, Kaichun; Liang, Jie

2017-12-20

Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis. Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd -/- ) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd -/- and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days. GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd -/- mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd -/- mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet. As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis

Fish oil feeding attenuates neuroinflammatory gene expression without concomitant changes in brain eicosanoids and docosanoids in a mouse model of Alzheimer's disease.

Science.gov (United States)

Hopperton, Kathryn E; Trépanier, Marc-Olivier; James, Nicholas C E; Chouinard-Watkins, Raphaël; Bazinet, Richard P

2018-03-01

Neuroinflammation is a recognized hallmark of Alzheimer's disease, along with accumulation of amyloid-β plaques, neurofibrillary tangles and synaptic loss. n-3 polyunsaturated fatty acids (PUFA) and molecules derived from them, including eicosapentaenoic acid-derived eicosanoids and docosahexaenoic acid-derived docosanoids, are known to have both anti-inflammatory and pro-resolving properties, while human observational data links consumption of these fatty acids to a decreased risk of Alzheimer's disease. Few studies have examined the neuroinflammation-modulating effects of n-3 PUFA feeding in an Alzheimer's disease-related model, and none have investigated whether these effects are mediated by changes in brain eicosanoids and docosanoids. Here, we use both a fat-1 transgenic mouse and a fish oil feeding model to study the impact of increasing tissue n-3 PUFA on neuroinflammation and the production of pro-inflammatory and pro-resolving lipid mediators. Fat-1 mice, transgenic animals that can convert n-6 to n-3 PUFA, and their wildtype littermates were fed diets containing either fish oil (high n-3 PUFA) or safflower oil (negligible n-3 PUFA) from weaning to 12 weeks. Animals then underwent intracerebroventricular infusion of either amyloid-β 1-40 or a control peptide. Hippocampi were collected from non-surgery and surgery animals 10 days after infusion. Microarray was used to measure enrichment of inflammation-associated gene categories and expression of genes involved in the synthesis of lipid mediators. Results were validated by real-time PCR in a separate cohort of animals. Lipid mediators were measured via liquid chromatography tandem mass spectrometry. Fat-1 and wildtype mice fed fish oil had higher total hippocampal DHA than wildtype mice fed the safflower oil diet. The safflower-fed mice, but not the fat-1 or fish oil-fed mice, had significantly increased expression in gene ontology categories associated with inflammation in response to amyloid

The glycolytic flux in Escherichia coli is controlled by the demand for ATP

DEFF Research Database (Denmark)

Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

2002-01-01

additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (> 75%) resides...... not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i...

Targeted Overexpression of Inducible 6-Phosphofructo-2-kinase in Adipose Tissue Increases Fat Deposition but Protects against Diet-induced Insulin Resistance and Inflammatory Responses*

Science.gov (United States)

Huo, Yuqing; Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Fan, Yang-Yi; Ong, Kuok Teong; Woo, Shih-Lung; Chapkin, Robert S.; Mashek, Douglas G.; Chen, Yanming; Dong, Hui; Lu, Fuer; Wei, Lai; Wu, Chaodong

2012-01-01

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues. PMID:22556414

Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

DEFF Research Database (Denmark)

Poulsen, Christian Bjørn; Penkowa, Milena; Borup, Rehannah

2005-01-01

Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays...... in wild-type and IL-6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post-lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with approximately 12,400 probe sets corresponding to approximately 10...... in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight...

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.

Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

International Nuclear Information System (INIS)

Seigne, Christelle; Fontanière, Sandra; Carreira, Christine; Lu, Jieli; Tong, Wei-Ming; Fontanière, Bernard; Wang, Zhao-Qi; Zhang, Chang Xian; Frappart, Lucien

2010-01-01

Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1 +/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Six Men1 +/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland

Mice deficient in cryptochrome 1 (Cry1-/- exhibit resistance to obesity induced by a high fat diet

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Guy eGriebel

2014-04-01

Full Text Available Disruption of circadian clock enhances the risk of metabolic syndrome, obesity, and type 2 diabetes. Circadian clocks rely on a highly regulated network of transcriptional and translational loops that drive clock-controlled gene expression. Among these transcribed clock genes are cryptochrome (CRY family members, which comprise Cry1 and Cry2. While the metabolic effects of deletion of several core components of the clock gene machinery have been well characterized, those of selective inactivation of Cry1 or Cry2 genes have not been described. In this study we demonstrate that ablation of Cry1, but not Cry2, prevents high-fat diet (HFD-induced obesity in mice. Despite similar caloric intake, Cry1-/- mice on HFD gained markedly less weight (-18 % at the end of the 16-week experiment and displayed reduced fat accumulation compared to wild-type (WT littermates (-61 %, suggesting increased energy expenditure. Analysis of serum lipid and glucose profiles showed no difference between Cry1-/- and WT mice. Both Cry1-/- and Cry2-/- mice are indistinguishable from WT controls in body weight, fat and protein contents, and food consumption when they are allowed unlimited access to a standard rodent diet. We conclude that although CRY signaling may not be essential for the maintenance of energy homeostasis under steady-state nutritional conditions, Cry1 may play a role in readjusting energy balance under changing nutritional circumstances. These studies reinforce the important role of circadian clock genes in energy homeostasis and suggest that Cry1 is a plausible target for antiobesity therapy.

Mice deficient in cryptochrome 1 (cry1 (-/-)) exhibit resistance to obesity induced by a high-fat diet.

Science.gov (United States)

Griebel, Guy; Ravinet-Trillou, Christine; Beeské, Sandra; Avenet, Patrick; Pichat, Philippe

2014-01-01

Disruption of circadian clock enhances the risk of metabolic syndrome, obesity, and type 2 diabetes. Circadian clocks rely on a highly regulated network of transcriptional and translational loops that drive clock-controlled gene expression. Among these transcribed clock genes are cryptochrome (CRY) family members, which comprise Cry1 and Cry2. While the metabolic effects of deletion of several core components of the clock gene machinery have been well characterized, those of selective inactivation of Cry1 or Cry2 genes have not been described. In this study, we demonstrate that ablation of Cry1, but not Cry2, prevents high-fat diet (HFD)-induced obesity in mice. Despite similar caloric intake, Cry1 (-/-) mice on HFD gained markedly less weight (-18%) at the end of the 16-week experiment and displayed reduced fat accumulation compared to wild-type (WT) littermates (-61%), suggesting increased energy expenditure. Analysis of serum lipid and glucose profiles showed no difference between Cry1 (-/-) and WT mice. Both Cry1 (-/-) and Cry2 (-/-) mice are indistinguishable from WT controls in body weight, fat and protein contents, and food consumption when they are allowed unlimited access to a standard rodent diet. We conclude that although CRY signaling may not be essential for the maintenance of energy homeostasis under steady-state nutritional conditions, Cry1 may play a role in readjusting energy balance under changing nutritional circumstances. These studies reinforce the important role of circadian clock genes in energy homeostasis and suggest that Cry1 is a plausible target for anti-obesity therapy.

Role of passive T-cell death in chronic experimental autoimmune encephalomyelitis

DEFF Research Database (Denmark)

Issazadeh-Navikas, Shohreh; Abdallah, K; Chitnis, T

2000-01-01

central nervous system (CNS) compared with controls. There was also a decreased number of apoptotic cells in the CNS of Bcl-x(L) transgenic mice when compared with littermates at all time points tested. This is the first report of an autoimmune disease model in Bcl-x(L) transgenic mice. Our data indicate...

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

Science.gov (United States)

Botes, D P; Qobose, M D; Corfield, V A

1991-09-15

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

Iron and zinc complexation in wild-type and ferritin-expressing wheat grain: implications for mineral transport into developing grain

DEFF Research Database (Denmark)

Neal, Andrew L; Geraki, Kalotina; Borg, Søren

2013-01-01

of modified complexation of both metals in transgenic grain overexpressing wheat ferritin. For zinc, there is a consistent doubling of the number of complexing phosphorus atoms. Although there is some EXAFS evidence for iron phytate in ferritin-expressing grain, there is also evidence of a structure lacking......We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells...... of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain....

Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis.

Science.gov (United States)

Wang, Yan; Quagliarini, Fabiana; Gusarova, Viktoria; Gromada, Jesper; Valenzuela, David M; Cohen, Jonathan C; Hobbs, Helen H

2013-10-01

Angiopoietin-like protein (ANGPTL)8 (alternatively called TD26, RIFL, Lipasin, and Betatrophin) is a newly recognized ANGPTL family member that has been implicated in both triglyceride (TG) and glucose metabolism. Hepatic overexpression of ANGPTL8 causes hypertriglyceridemia and increased insulin secretion. Here we examined the effects of inactivating Angptl8 on TG and glucose metabolism in mice. Angptl8 knockout (Angptl8(-/-)) mice gained weight more slowly than wild-type littermates due to a selective reduction in adipose tissue accretion. Plasma levels of TGs of the Angptl8(-/-) mice were similar to wild-type animals in the fasted state but paradoxically decreased after refeeding. The lower TG levels were associated with both a reduction in very low density lipoprotein secretion and an increase in lipoprotein lipase (LPL) activity. Despite the increase in LPL activity, the uptake of very low density lipoprotein-TG is markedly reduced in adipose tissue but preserved in hearts of fed Angptl8(-/-) mice. Taken together, these data indicate that ANGPTL8 plays a key role in the metabolic transition between fasting and refeeding; it is required to direct fatty acids to adipose tissue for storage in the fed state. Finally, glucose and insulin tolerance testing revealed no alterations in glucose homeostasis in mice fed either a chow or high fat diet. Thus, although absence of ANGPTL8 profoundly disrupts TG metabolism, we found no evidence that it is required for maintenance of glucose homeostasis.

Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory.

Science.gov (United States)

Bradley, Ryan M; Mardian, Emily B; Bloemberg, Darin; Aristizabal Henao, Juan J; Mitchell, Andrew S; Marvyn, Phillip M; Moes, Katherine A; Stark, Ken D; Quadrilatero, Joe; Duncan, Robin E

2017-11-15

We previously characterized LPAATδ/AGPAT4 as a mitochondrial lysophosphatidic acid acyltransferase that regulates brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Here, we report that Lpaat δ -/- mice display impaired spatial learning and memory compared to wild-type littermates in the Morris water maze and our investigation of potential mechanisms associated with brain phospholipid changes. Marker protein immunoblotting suggested that the relative brain content of neurons, glia, and oligodendrocytes was unchanged. Relative abundance of the important brain fatty acid docosahexaenoic acid was also unchanged in phosphatidylserine, phosphatidylglycerol, and cardiolipin, in agreement with prior data on PC, PE and PI. In phosphatidic acid, it was increased. Specific decreases in ethanolamine-containing phospholipids were detected in mitochondrial lipids, but the function of brain mitochondria in Lpaat δ -/- mice was unchanged. Importantly, we found that Lpaat δ -/- mice have a significantly and drastically lower brain content of the N -methyl-d-asparate (NMDA) receptor subunits NR1, NR2A, and NR2B, as well as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1, compared to wild-type mice. However, general dysregulation of PI-mediated signaling is not likely responsible, since phospho-AKT and phospho-mTOR pathway regulation was unaffected. Our findings indicate that Lpaat δ deficiency causes deficits in learning and memory associated with reduced NMDA and AMPA receptors. Copyright © 2017 American Society for Microbiology.

Bioconversion Studies of Methyl Laurate to Dodecanedioic Acid using a Wild-type of Candida tropicalis

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Akmalina Rifkah

2018-01-01

Full Text Available Production of dodecanedioic acid (DDDA, a platform chemical used as raw material for various commodities and polymers, has been studied through a biological process. This process was conducted by using a wild-type of Candida tropicalis which can be obtained easily from natural resources. The aim of this research was to study the characteristics of DDDA production from methyl laurate through batch fermentation process. Growth phase was carried out for 20 h, as the beginning of fermentation, then continued to conversion phase for 5 until 6 days. Utilization of methyl laurate and production of DDDA were analysed using gas chromatography, which proved the ability of C. tropicalis in assimilating methyl laurate to convert it become DDDA. The highest value of cells yield (Yx/s and DDDA yield (Yp/s successfully obtained were 0.86 g cells/g methyl laurate and 0.20 g DDDA/g methyl laurate, respectively. This study also showed the possibility of fermentation products accumulation as intermediate, or accumulation of DDDA inside the cells. Thus, this study can be applied as an alternative in addition to the use of mutant microorganism in producing DDDA.

SAD-B kinase regulates pre-synaptic vesicular dynamics at hippocampal Schaffer collateral synapses and affects contextual fear memory.

Science.gov (United States)

Watabe, Ayako M; Nagase, Masashi; Hagiwara, Akari; Hida, Yamato; Tsuji, Megumi; Ochiai, Toshitaka; Kato, Fusao; Ohtsuka, Toshihisa

2016-01-01

Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. © 2015 International

Neonatal oxytocin and vasopressin manipulation alter social behavior during the juvenile period in Mongolian gerbils.

Science.gov (United States)

Taylor, Jack H; Cavanaugh, Jon; French, Jeffrey A

2017-07-01

Oxytocin and vasopressin are important modulators of a wide variety of social behaviors, and increasing evidence is showing that these neuropeptides are important organizational effectors of later-life behavior as well. We treated day-old gerbil pups with oxytocin, vasopressin, an oxytocin receptor antagonist, a vasopressin V1a receptor antagonist, or saline control, and then measured received parental responsiveness during the early postnatal period and juvenile social behavior during weaning. Neonatal vasopressin treatment enhanced sociality in males, but not females, at both developmental time points. When pups were individually placed outside the nest, parents were more responsive to male pups treated with vasopressin compared with littermates, and vasopressin treated male pups exhibited increased play with littermates as juveniles. These results show that vasopressin during very early life can enhance social interactions throughout early development. © 2017 Wiley Periodicals, Inc.

Olfactory granule cell development in normal and hyperthyroid rats.

Science.gov (United States)

Brunjes, P C; Schwark, H D; Greenough, W T

1982-10-01

Dendritic development was examined in olfactory bulbs of both normal 7-, 14-, 21- and 60-day-old rats and littermates treated on postnatal days 1-4 with 1 microgram/g body weight of L-thyroxine sodium. Tissue was processed via the Golgi-Cox technique and subjected to quantitative analyses of mitral and internal layer granule cell development. These populations of granule cells were selected because their pattern of late proliferation suggested potentially greater susceptibility to postnatal hormonal alterations. Although neonatal hyperthyroidism induces widespread acceleration of maturation, including precocious chemosensitivity, granule cell development was unaffected relative to littermate controls. Both normal and hyperthyroid groups exhibited an inverted U-shaped pattern of cellular development, with rapid dendritic dendritic growth and expansion occurring during the earliest ages tested, but with loss of processes and dendritic field size occurring after day 21.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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Cavazzoni Andrea

2012-12-01

Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

Science.gov (United States)

Clerc, Jérôme; Florea, Bogdan I; Kraus, Marianne; Groll, Michael; Huber, Robert; Bachmann, André S; Dudler, Robert; Driessen, Christoph; Overkleeft, Herman S; Kaiser, Markus

2009-11-02

The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

Science.gov (United States)

1996-08-01

that my statement of work (SOW) for the current project omitted many of the tasks that had to be carried out in order to get the lab up and running...that we knew that we could stabilize wild-type p53 in ML-1 cells along with the possibility of being able to get an excellent elutriation profile with...nuclear protein extract was immunoprecipitated with PAb421 cross-linked to ProteinA -Sepharose beads and analysed by SDS-PAGE Western blot analysis with

Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

Directory of Open Access Journals (Sweden)

Yang Moon-Sik

2004-09-01

Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

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Scartozzi Mario

2009-08-01

Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

NF-kappaB Activity in Macrophages Determines Metastatic Potential of Breast Tumor Cells

Science.gov (United States)

2011-08-01

about the relatively low number of mice in the control group so we decided to generate additional mice to increase the numbers in each group (Second...mammary epithelium increases tumor latency and decreases tumor burden. Oncogene 2010, In press. 10. Connelly L, Barham W, Pigg R, Saint-Jean L, Sherrill T...littermate controls (Fig. 5E, 5G ). These differences were consistent with defects in saccular airway branching morphogenesis. The lack of effect in mice

Intestine-specific overexpression of IL-10 improves survival in polymicrobial sepsis.

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Rajan, Saju; Vyas, Dinesh; Clark, Andrew T; Woolsey, Cheryl A; Clark, Jessica A; Hotchkiss, Richard S; Buchman, Timothy G; Coopersmith, Craig M

2008-04-01

Targeted IL-10 therapy improves survival in preclinical models of critical illness, and intestine-specific IL-10 decreases inflammation in models of chronic Inflammatory disease. We therefore sought to determine whether intestine-specific overexpression of IL-10 would improve survival in sepsis. Transgenic mice that overexpress IL-10 in their gut epithelium (Fabpi-IL-10 mice) and wild-type (WT) littermates (n = 127) were subjected to cecal ligation and puncture with a 27-gauge needle. The 7-day survival rate was 45% in transgenic animals and 30% in WT animals (P < or = 0.05). Systemic levels of IL-10 were undetectable in both groups of animals under basal conditions and were elevated to a similar degree in septic animals regardless of whether they expressed the transgene. Local parameter of injury, including gut epithelial apoptosis, intestinal permeability, peritoneal lavage cytokines, and stimulated cytokines from intraepithelial lymphocytes, were similar between transgenic and WT mice. However, in stimulated splenocytes, proinflammatory cytokines monocyte chemoattractant protein 1 (189 +/- 43 vs. 40 +/- 8 pg/mL) and IL-6 (116 +/- 28 vs. 34 +/- 9 pg/mL) were lower in Fabpi-IL-10 mice than WT littermates despite the intestine-specific nature of the transgene (P < 0.05). Cytokine levels were similar in blood and bronchoalveolar lavage fluid between the 2 groups, as were circulating LPS levels. Transgenic mice also had lower white blood cell counts associated with lower absolute neutrophil counts (0.5 +/- 0.1 vs. 1.0 +/- 0.2 10(3)/mm3; P < 0.05). These results indicate that gut-specific overexpression of IL-10 improves survival in a murine model of sepsis, and interactions between the intestinal epithelium and the systemic immune system may play a role in conferring this survival advantage.

CB1 receptor-mediated signaling underlies the hippocampal synaptic, learning, and memory deficits following treatment with JWH-081, a new component of spice/K2 preparations.

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Basavarajappa, Balapal S; Subbanna, Shivakumar

2014-02-01

Recently, synthetic cannabinoids have been sprayed onto plant material, which is subsequently packaged and sold as "Spice" or "K2" to mimic the effects of marijuana. A recent report identified several synthetic additives in samples of "Spice/K2", including JWH-081, a synthetic ligand for the cannabinoid receptor 1 (CB1). The deleterious effects of JWH-081 on brain function are not known, particularly on CB1 signaling, synaptic plasticity, learning and memory. Here, we evaluated the effects of JWH-081 on pCaMKIV, pCREB, and pERK1/2 signaling events followed by long-term potentiation (LTP), hippocampal-dependent learning and memory tasks using CB1 receptor wild-type (WT) and knockout (KO) mice. Acute administration of JWH-081 impaired CaMKIV phosphorylation in a dose-dependent manner, whereas inhibition of CREB phosphorylation in CB1 receptor WT mice was observed only at higher dose of JWH-081 (1.25 mg/kg). JWH-081 at higher dose impaired CaMKIV and CREB phosphorylation in a time-dependent manner in CB1 receptor WT mice but not in KO mice and failed to alter ERK1/2 phosphorylation. In addition, SR treated or CB1 receptor KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio compared with vehicle or WT littermates. In hippocampal slices, JWH-081 impaired LTP in CB1 receptor WT but not in KO littermates. Furthermore, JWH-081 at higher dose impaired object recognition, spontaneous alternation and spatial memory on the Y-maze in CB1 receptor WT mice but not in KO mice. Collectively our findings suggest that deleterious effects of JWH-081 on hippocampal function involves CB1 receptor mediated impairments in CaMKIV and CREB phosphorylation, LTP, learning and memory in mice. © 2013 Wiley Periodicals, Inc.

Elevation of endogenous anandamide impairs LTP, learning, and memory through CB1 receptor signaling in mice.

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Basavarajappa, Balapal S; Nagre, Nagaraja N; Xie, Shan; Subbanna, Shivakumar

2014-07-01

In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses. © 2014 Wiley Periodicals, Inc.

Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

OpenAIRE

2013-01-01

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concom...

Using ICP and micro-PIXE to investigate possible differences in the mineral composition of genetically modified versus wild-type sorghum grain

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Ndimba, R.; Cloete, K.; Mehlo, L.; Kossmann, J.; Mtshali, C.; Pineda-Vargas, C.

2017-08-01

In the present study, possible differences in the mineral composition of transgenic versus non-transgenic sorghum grains were investigated using inductively coupled atomic emission spectroscopy (ICP-AES); and, in-tissue elemental mapping by micro Proton-Induced X-ray Emission (micro-PIXE) analysis. ICP AES was used to analyse the bulk mineral content of the wholegrain flour derived from each genotype; whilst micro-PIXE was used to interrogate localised differences in mineral composition specific to certain areas of the grain, such as the bran layer and the central endosperm tissue. According to the results obtained, no significant difference in the average Fe, Zn or Ca content was found to differentiate the transgenic from the wild-type grain using ICP-AES. However, using micro-PIXE, a significant reduction in zinc could be detected in the bran layer of the transgenic grains relative to wild-type. Although it is difficult to draw firm conclusions, as a result of the small sample size used in this study, micro-PIXE has nonetheless proven itself as a useful technique for highlighting the possibility that there may be reduced levels of zinc accumulation in the bran layer of the transgenic grains. Given that the genetic modification targets proteins that are highly concentrated in certain parts of the bran tissue, it seems plausible that the reduced levels of zinc may be an unintended consequence of the silencing of kafirin proteins. Although no immediate health or nutritional concerns emerge from this preliminary finding, it is noted that zinc plays an important biological role within this part of the grain as a structural stabiliser and antioxidant factor. Further study is therefore needed to assess more definitively the extent of the apparent localised reduction in zinc in the transgenic grains and how this may affect other important grain quality characteristics.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

Science.gov (United States)

Munhenga, Givemore; Brooke, Basil D; Gilles, Jeremie R L; Slabbert, Kobus; Kemp, Alan; Dandalo, Leonard C; Wood, Oliver R; Lobb, Leanne N; Govender, Danny; Renke, Marius; Koekemoer, Lizette L

2016-03-02

Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the

Expression of Cathepsins B, D, and G in Isocitrate Dehydrogenase-Wildtype Glioblastoma

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Sabrina P. Koh

2017-05-01

Full Text Available AimTo investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC subpopulations, we have previously characterized within isocitrate dehydogenase (IDH-wildtype glioblastoma (IDHWGB.Methods3,3-Diaminobezidine (DAB immunohistochemical (IHC staining for cathepsins B, D, and G, was performed on 4μm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric in situ hybridization (CISH were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance.ResultsCathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4+ and SALL4+ CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature.ConclusionThis study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more

Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

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Hogarth, Marshall W; Garton, Fleur C; Houweling, Peter J; Tukiainen, Taru; Lek, Monkol; Macarthur, Daniel G; Seto, Jane T; Quinlan, Kate G R; Yang, Nan; Head, Stewart I; North, Kathryn N

2016-03-01

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO2.

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Occhialini, Alessandro; Lin, Myat T; Andralojc, P John; Hanson, Maureen R; Parry, Martin A J

2016-01-01

Introducing a carbon-concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO2 fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild-type and required supplementary CO2 . Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild-type growth rates, although still requiring elevated CO2 . We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non-native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen-use efficiency that may be achieved provided that adequate CO2 is available near the enzyme. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Melatonin enhances cold tolerance in drought-primed wild-type and abscisic acid-deficient mutant barley.

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Li, Xiangnan; Tan, Dun-Xian; Jiang, Dong; Liu, Fulai

2016-10-01

Melatonin is involved in multiple plant developmental processes and various stress responses. To explore the roles of melatonin played as well as its association with abscisic acid (ABA) in a process of drought priming-induced cold tolerance (DPICT), a wild-type barley and its ABA-deficient mutant Az34 counterpart were selected for comparison, in which the effects of melatonin application (either foliarly or rhizospherically) and/or drought priming on the cold tolerance of both types of barleys were systematically investigated. It was demonstrated that the early drought priming induced an increase of endogenous melatonin production, which is not ABA dependent. In addition, exogenously applied melatonin resulted in higher ABA concentration in the drought-primed plants than in the nonprimed plants when exposed to cold stress, indicating that ABA responded in a drought-dependent manner. The interplay of melatonin and ABA leads to plants maintaining better water status. Drought priming-induced melatonin accumulation enhanced the antioxidant capacity in both chloroplasts and mitochondria, which sustained the photosynthetic electron transport in photosynthetic apparatus of the plants under cold stress. These results suggest that the exogenous melatonin application enhances the DPICT by modulating subcellular antioxidant systems and ABA levels in barley. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

Science.gov (United States)

Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

Kidney branching morphogenesis under the control of a ligand–receptor-based Turing mechanism

International Nuclear Information System (INIS)

Menshykau, Denis; Iber, Dagmar

2013-01-01

The main signalling proteins that control early kidney branching have been defined. Yet the underlying mechanism is still elusive. We have previously shown that a Schnakenberg-type Turing mechanism can recapitulate the branching and protein expression patterns in wild-type and mutant lungs, but it is unclear whether this mechanism would extend to other branched organs that are regulated by other proteins. Here, we show that the glial cell line-derived neurotrophic factor–RET regulatory interaction gives rise to a Schnakenberg-type Turing model that reproduces the observed budding of the ureteric bud from the Wolffian duct, its invasion into the mesenchyme and the observed branching pattern. The model also recapitulates all relevant protein expression patterns in wild-type and mutant mice. The lung and kidney models are both based on a particular receptor–ligand interaction and require (1) cooperative binding of ligand and receptor, (2) a lower diffusion coefficient for the receptor than for the ligand and (3) an increase in the receptor concentration in response to receptor–ligand binding (by enhanced transcription, more recycling or similar). These conditions are met also by other receptor–ligand systems. We propose that ligand–receptor-based Turing patterns represent a general mechanism to control branching morphogenesis and other developmental processes. (paper)

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.

Altered metabolic incorporation of fucose and leucine into PNS myelin of 25-week-old diabetic (C57BL/Ks [db/db]) mice: effects of untreated diabetes on nerve metabolism

International Nuclear Information System (INIS)

Chez, M.G.; Peterson, R.G.

1983-01-01

Sciatic nerves of 25-week-old genetically diabetic (C57BL/Ks [db/db]) mice and their litter-mate controls were removed, and their metabolic incorporation of [ 3 H]fucose and [ 14 C]leucine into myelin was studied in vitro. Untreated diabetic animals showed significant increases (p less than 0.05) in the fucose/leucine incorporation into myelin when compared to values found for their litter-mates. These results correlated well with previous experiments performed on alloxan or streptozotocin-diabetic rats and thus show the in vitro incubation procedure to be a good indicator of altered metabolic conditions in peripheral nerves due to diabetes mellitus. The resulting ratio increases seen in diabetic animals is at variance with the decrease in ratios found in animals undergoing typical Wallerian degeneration. These results suggest that different metabolic processes operate in untreated diabetics than in normals or in those undergoing other degenerative nerve processes

Administration of soluble activin receptor 2B increases bone and muscle mass in a mouse model of osteogenesis imperfecta

Science.gov (United States)

DiGirolamo, Douglas J.; Singhal, Vandana; Chang, Xiaoli; Lee, Se-Jin; Germain-Lee, Emily L.

2015-01-01

Osteogenesis imperfecta (OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI, many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B (ACVR2B) in a mouse model of type III OI (oim). Treatment of 12-week-old oim mice with ACVR2B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy, wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system. PMID:26161291

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

Science.gov (United States)

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

2008-09-01

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

Hearing dysfunction in heterozygous Mitf(Mi-wh) /+ mice, a model for Waardenburg syndrome type 2 and Tietz syndrome.

Science.gov (United States)

Ni, Christina; Zhang, Deming; Beyer, Lisa A; Halsey, Karin E; Fukui, Hideto; Raphael, Yehoash; Dolan, David F; Hornyak, Thomas J

2013-01-01

The human deafness-pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia-White (Mitf(Mi-wh) /+) mice were studied and hearing function of these mice characterized. Mitf(Mi-wh) /+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf(Mi-wh) /+ embryos have fewer melanoblasts during embryonic development than their wild-type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf(Mi-wh) /+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness-pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes. © 2012 John Wiley & Sons A/S.

Secretoglobin Superfamily Protein SCGB3A2 Deficiency Potentiates Ovalbumin-Induced Allergic Pulmonary Inflammation

Directory of Open Access Journals (Sweden)

Taketomo Kido

2014-01-01

Full Text Available Secretoglobin (SCGB 3A2, a cytokine-like secretory protein of small molecular weight, which may play a role in lung inflammation, is predominantly expressed in airway epithelial cells. In order to understand the physiological role of SCGB3A2, Scgb3a2−/− mice were generated and characterized. Scgb3a2−/− mice did not exhibit any overt phenotypes. In ovalbumin- (OVA- induced airway allergy inflammation model, Scgb3a2−/− mice in mixed background showed a decreased OVA-induced airway inflammation, while six times C57BL/6NCr backcrossed congenic Scgb3a2−/− mice showed a slight exacerbation of OVA-induced airway inflammation as compared to wild-type littermates. These results indicate that the loss of SCGB3A2 function was influenced by a modifier gene(s in mixed genetic background and suggest that SCGB3A2 has anti-inflammatory property. The results further suggest the possible use of recombinant human SCGB3A2 as an anti-inflammatory agent.

Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice

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Peng Zhang

2016-06-01

Full Text Available De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs compared with wild-type littermates. Asxl1−/− BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1−/− BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development.

Robust GLP-1 secretion by basic L-amino acids does not require the GPRC6A receptor

DEFF Research Database (Denmark)

Clemmensen, Christoffer; Jørgensen, Christinna V; Smajilovic, Sanela

2017-01-01

(GLP-1) secretion is unclear. Therefore, to probe if the GPRC6A receptor is indispensible for amino acid-induced secretion of GLP-1, we treated, with oral gavage, GPRC6A knock-out (KO) and wild-type (WT) littermate mice with GPRC6A ligands: L-arginine and L-ornithine, and assessed GLP-1 levels...... in circulation. We found that oral administration of both L-arginine and L-ornithine significantly increased total plasma GLP-1 levels to a similar level in GPRC6A KO and WT mice 15 minutes after gavage (both amino acids) and accumulated up to 60 minutes after gavage (L-arginine). Conversely, GLP-1 secretion...... at the 30 and 60 minute time points in the KO mice were attenuated and did not reach statistical significance. In summary, these data confirm that L-arginine is a potent GLP-1 secretagogue and show that the main effect occurs independently of GPRC6A. In addition, this is the first study to show that also L...

The mother or the fetus? 11beta-hydroxysteroid dehydrogenase type 2 null mice provide evidence for direct fetal programming of behavior by endogenous glucocorticoids.

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Holmes, Megan C; Abrahamsen, Christian T; French, Karen L; Paterson, Janice M; Mullins, John J; Seckl, Jonathan R

2006-04-05

Low birth weight associates with increased susceptibility to adult cardiometabolic and affective disorders spawning the notion of fetal "programming." Prenatal exposure to excess glucocorticoids may be causal. In support, maternal stress or treatment during pregnancy with dexamethasone (which crosses the placenta) or inhibitors of fetoplacental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the physiological "barrier" to maternal glucocorticoids, reduces birth weight and programs permanent offspring hypertension, hyperglycemia, and anxiety behaviors. It remains uncertain whether such effects are mediated indirectly via altered maternal function or directly on the fetus and its placenta. To dissect this critical issue, we mated 11beta-HSD2(+/-) mice such that each pregnant female produces +/+, +/-, and -/- offspring and compared them with offspring of homozygous wild-type and -/- matings. We show that 11beta-HSD2(-/-) offspring of either +/- or -/- mothers have lower birth weight and exhibit greater anxiety than 11beta-HSD2(+/+) littermates. This provides clear evidence for the key role of fetoplacental 11beta-HSD2 in prenatal glucocorticoid programming.

Cognitive abilities of Alzheimer's disease transgenic mice are modulated by social context and circadian rhythm.

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Kiryk, Anna; Mochol, Gabriela; Filipkowski, Robert K; Wawrzyniak, Marcin; Lioudyno, Victoria; Knapska, Ewelina; Gorkiewicz, Tomasz; Balcerzyk, Marcin; Leski, Szymon; Leuven, Fred Van; Lipp, Hans-Peter; Wojcik, Daniel K; Kaczmarek, Leszek

2011-12-01

In the present study, we used a new training paradigm in the intelliCage automatic behavioral assessment system to investigate cognitive functions of the transgenic mice harboring London mutation of the human amyloid precursor protein (APP.V717I). Three groups of animals: 5-, 12- and 18-24-month old were subjected to both Water Maze training and the IntelliCage-based appetitive conditioning. The spatial memory deficit was observed in all three groups of transgenic mice in both behavioral paradigms. However, the APP mice were capable to learn normally when co-housed with the wild-type (WT) littermates, in contrast to clearly impaired learning observed when the transgenic mice were housed alone. Furthermore, in the transgenic mice kept in the Intellicage alone, the cognitive deficit of the young animals was modulated by the circadian rhythm, namely was prominent only during the active phase of the day. The novel approach to study the transgenic mice cognitive abilities presented in this paper offers new insight into cognitive dysfunctions of the Alzheimer's disease mouse model.

Structural alterations in tumor-draining lymph nodes before papillary thyroid carcinoma metastasis.

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Hinson, Andrew M; Massoll, Nicole A; Jolly, Lee Ann; Stack, Brendan C; Bodenner, Donald L; Franco, Aime T

2017-08-01

The purpose of this study was to define and characterize the thyroid tumor-draining lymph nodes in genetically engineered mice harboring thyroid-specific expression of oncogenic Braf V600E with and without Pten insufficiency. After intratumoral injection of methylene blue, the lymphatic drainage of the thyroid gland was visualized in real time. The thyroid gland/tumor was resected en bloc with the respiratory system for histological analysis. Although mice harboring Braf V600E mutations were smaller in body size compared with their wild-type (WT) littermates, the size of their thyroid glands and deep cervical lymph nodes were significantly larger. Additionally, the tumor-draining lymph nodes showed increased and enlarged lymphatic sinuses that were distributed throughout the cortex and medulla. Tumor-reactive lymphadenopathy and histiocytosis, but no frank metastases, were observed in all mice harboring Braf V600E mutations. The tumor-draining lymph nodes undergo significant structural alterations in immunocompetent mice, and this may represent a primer for papillary thyroid carcinoma (PTC) metastasis. © 2017 Wiley Periodicals, Inc.

Variant BDNF-Val66Met Polymorphism is Associated with Layer-Specific Alterations in GABAergic Innervation of Pyramidal Neurons, Elevated Anxiety and Reduced Vulnerability of Adolescent Male Mice to Activity-Based Anorexia.

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Chen, Yi-Wen; Surgent, Olivia; Rana, Barkha S; Lee, Francis; Aoki, Chiye

2017-08-01

Previously, we determined that rodents' vulnerability to food restriction (FR)-evoked wheel running during adolescence (activity-based anorexia, ABA) is associated with failures to increase GABAergic innervation of hippocampal and medial prefrontal pyramidal neurons. Since brain-derived neurotrophic factor (BDNF) promotes GABAergic synaptogenesis, we hypothesized that individual differences in this vulnerability may arise from differences in the link between BDNF bioavailability and FR-evoked wheel running. We tested this hypothesis in male BDNF-Val66Met knock-in mice (BDNFMet/Met), known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. We found that 1) in the absence of FR or a wheel (i.e., control), BDNFMet/Met mice are more anxious than wild-type (WT) littermates, 2) electron microscopically verified GABAergic innervations of pyramidal neurons of BDNFMet/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, 3) following ABA, WT mice exhibit anxiety equal to those of the BDNFMet/Met mice and have lost GABAergic innervation along distal dendrites, 4) BDNFMet/Met mice show blunted ABA vulnerability, and 5) unexpectedly, GABAergic innervation is higher at somata of BDNFMet/Met mice than of WT. We conclude that lamina-specific GABAergic inhibition is important for regulating anxiety, whether arising from environmental stress, such as food deprivation, or genetically, such as BDNFMet/Met single nucleotide polymorphism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice.

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Fazal, Loubina; Azibani, Feriel; Bihry, Nicolas; Coutance, Guillaume; Polidano, Evelyne; Merval, Régine; Vodovar, Nicolas; Launay, Jean-Marie; Delcayre, Claude; Samuel, Jane-Lise

2014-06-01

Studies have shown that aldosterone would have angiogenic effects and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes and the molecular pathways involved. Male 3-wk-old aldosterone synthase (AS)-overexpressing mice and their control wild-type (WT) littermates were fed a standard or high-fat, high-sucrose (HFHS) diet. After 6 mo of diet treatment, mice were euthanized, and cardiac samples were assayed by RT-PCR, immunoblotting, and immunohistology. HFHS diet induced type-2 diabetes in WT (WT-D) and AS (AS-D) mice. VEGFa mRNAs decreased in WT-D (-43%, P<0.05 vs. WT) and increased in AS-D mice (+236%, P< 0.01 vs. WT-D). In WT-D mouse hearts, the proapoptotic p38MAPK was activated (P<0.05 vs. WT and AS-D), whereas Akt activity decreased (-64%, P<0.05 vs. WT). The AS mice, which exhibited a cardiac up-regulation of IGF1-R, showed an increase in Akt phosphorylation when diabetes was induced (P<0.05 vs. WT and AS-D). Contrary to WT-D mice, AS-D mouse hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D). To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders. © FASEB.

Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance

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Liu, Meilian; Xiang, Ruihua; Wilk, Sarah Ann; Zhang, Ning; Sloane, Lauren B.; Azarnoush, Kian; Zhou, Lijun; Chen, Hongzhi; Xiang, Guangda; Walter, Christi A.; Austad, Steven N.; Musi, Nicolas; DeFronzo, Ralph A.; Asmis, Reto; Scherer, Philipp E.; Dong, Lily Q.; Liu, Feng

2012-01-01

The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad−/−). In addition, the fDsbA-L/Ad−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders. PMID:22807031

Intravitreal administration of HA-1077, a ROCK inhibitor, improves retinal function in a mouse model of huntington disease.

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Mei Li

Full Text Available Huntington disease (HD is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt. The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK, which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

Attenuation of the progression of articular cartilage degeneration by inhibition of TGF-β1 signaling in a mouse model of osteoarthritis.

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Chen, Rebecca; Mian, Michelle; Fu, Martin; Zhao, Jing Ying; Yang, Liang; Li, Yefu; Xu, Lin

2015-11-01

Transforming growth factor beta 1 (TGF-β1) is implicated in osteoarthritis. We therefore studied the role of TGF-β1 signaling in the development of osteoarthritis in a developmental stage-dependent manner. Three different mouse models were investigated. First, the Tgf-β receptor II (Tgfbr2) was specifically removed from the mature cartilage of joints. Tgfbr2-deficient mice were grown to 12 months of age and were then euthanized for collection of knee and temporomandibular joints. Second, Tgfbr2-deficient mice were subjected to destabilization of the medial meniscus (DMM) surgery. Knee joints were then collected from the mice at 8 and 16 weeks after the surgery. Third, wild-type mice were subjected to DMM at the age of 8 weeks. Immediately after the surgery, these mice were treated with the Tgfbr2 inhibitor losartan for 8 weeks and then euthanized for collection of knee joints. All joints were characterized for evidences of articular cartilage degeneration. Initiation or acceleration of articular cartilage degeneration was not observed by the genetic inactivation of Tgfbr2 in the joints at the age of 12 months. In fact, the removal of Tgfbr2 and treatment with losartan both delayed the progression of articular cartilage degeneration induced by DMM compared with control littermates. Therefore, we conclude that inhibition of Tgf-β1 signaling protects adult knee joints in mice against the development of osteoarthritis. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Social approach behaviors are similar on conventional versus reverse lighting cycles, and in replications across cohorts, in BTBR T+ tf/J, C57BL/6J, and vasopressin receptor 1B mutant mice

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Mu Yang

2007-11-01

Full Text Available Mice are a nocturnal species, whose social behaviors occur primarily during the dark phase of the circadian cycle. However, laboratory rodents are frequently tested during their light phase, for practical reasons. We investigated the question of whether light phase testing presents a methodological pitfall for investigating mouse social approach behaviors. Three lines of mice were systematically compared. One cohort of each line was raised in a conventional lighting schedule and tested during the light phase, under white light illumination; another cohort was raised in a reverse lighting schedule and tested during their dark phase, under dim red light. Male C57BL/6J (B6 displayed high levels of sociability in our three-chambered automated social approach task when tested in either phase. BTBR T+ tf/J (BTBR displayed low levels of sociability in either phase. Five cohorts of vasopressin receptor subtype 1b (Avpr1b null mutants, heterozygotes, and wildtype littermate controls were tested in the same social approach paradigm: three in the dark phase and two in the light phase. All three genotypes displayed normal sociability in four out of the five replications. In the juvenile play test, testing phase had no effect on play soliciting behaviors in Avpr1b mice, but had modest effects on nose sniff and huddling. Taken together, these findings indicate that testing phase is not a crucial factor for studying some forms of social approach in juvenile and adult mice.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

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Samantha L Eaton

Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

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Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

2015-11-01

Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus

International Nuclear Information System (INIS)

Meier, P.; Scougall, C.A.; Will, H.; Burrell, C.J.; Jilbert, A.R.

2003-01-01

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i) their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii) the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii) the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV

Shared effects of genetic and intrauterine and perinatal environment on the development of metabolic syndrome.

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Patricia M Vuguin

Full Text Available Genetic and environmental factors, including the in utero environment, contribute to Metabolic Syndrome. Exposure to high fat diet exposure in utero and lactation increases incidence of Metabolic Syndrome in offspring. Using GLUT4 heterozygous (G4+/- mice, genetically predisposed to Type 2 Diabetes Mellitus, and wild-type littermates we demonstrate genotype specific differences to high fat in utero and lactation. High fat in utero and lactation increased adiposity and impaired insulin and glucose tolerance in both genotypes. High fat wild type offspring had increased serum glucose and PAI-1 levels and decreased adiponectin at 6 wks of age compared to control wild type. High fat G4+/- offspring had increased systolic blood pressure at 13 wks of age compared to all other groups. Potential fetal origins of adult Metabolic Syndrome were investigated. Regardless of genotype, high fat in utero decreased fetal weight and crown rump length at embryonic day 18.5 compared to control. Hepatic expression of genes involved in glycolysis, gluconeogenesis, oxidative stress and inflammation were increased with high fat in utero. Fetal serum glucose levels were decreased in high fat G4+/- compared to high fat wild type fetuses. High fat G4+/-, but not high fat wild type fetuses, had increased levels of serum cytokines (IFN-γ, MCP-1, RANTES and M-CSF compared to control. This data demonstrates that high fat during pregnancy and lactation increases Metabolic Syndrome male offspring and that heterozygous deletion of GLUT4 augments susceptibility to increased systolic blood pressure. Fetal adaptations to high fat in utero that may predispose to Metabolic Syndrome in adulthood include changes in fetal hepatic gene expression and alterations in circulating cytokines. These results suggest that the interaction between in utero-perinatal environment and genotype plays a critical role in the developmental origin of health and disease.

Comparison of genome engineering using the CRISPR-Cas9 system in C. glabrata wild-type and lig4 strains.

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Cen, Yuke; Timmermans, Bea; Souffriau, Ben; Thevelein, Johan M; Van Dijck, Patrick

2017-10-01

Candida glabrata is reported as the second most prevalent human opportunistic fungal pathogen in North America and is threatening patients all over the world. Its incidence is rising, while it has developed resistance to the most widely used antifungal drugs, necessitating new approaches based on better insight into the biology of the organism. Despite its close phylogenetic relationship with Saccharomyces cerevisiae, generating precise genomic alterations in this species is problematic. Previously we have shown that deletion of LIG4, which encodes an enzyme involved in Non-Homologous End Joining (NHEJ), strongly enhances the probability of obtaining correctly modified transformants. In this work we used the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system to genetically engineer the C. glabrata genome, targeting the genes ADE2, MET15 and SOK2, located on different chromosomes. We used the CRISPR-Cas9 technology to replace the open reading frame (ORF) by the SAT1 selective marker or introduced a premature stop codon in ADE2 and MET15, as they are easily scored by their adenine or methionine auxotrophy, respectively. The SOK2 gene was modified by insertion of a triple HA-tag sequence and the transformants were verified in a western blot. The CRISPR-Cas9 mediated targeting efficiency varies depending on the gene targeted and the genetic modification performed. We show that CRISPR-Cas9 mediated genome editing is more efficient than the conventional method in the wild-type strain, moreover it has the big advantage being marker-free. In previous work, we showed that the targeting efficiency is highly increased in the lig4Δ strain using the conventional way to delete genes in C. glabrata. Using the CRISPR-Cas9 system in this strain, the percentage of correct transformants is consistently higher compared to the wild-type strain. This indicates that using the lig4 mutant as such is already a strong

Rearrangement of RAG-1 recombinase gene in radiation-sensitive ''wasted'' mice

International Nuclear Information System (INIS)

Woloschak, G.E.; Weaver, P.

1994-01-01

The recent cloning and characterization of recombinase genes (RAG- 1/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice (wst). Our results revealed expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/sm-bullet mice). In thymus tissue, a small RAG-1 transcript was detected in wst/wst mice that was not evident in thymus from control mice. In wst/lg-bullet mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/sm-bullet and not from wst;/wst or parental control BCF 1 mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage

Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.

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Khalid Arhzaouy

Full Text Available Heterozygous mutations in the human VCP (p97 gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia, ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia and HSP (hereditary spastic paraplegia. Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97 or mutant p97 (p97(R155C fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO cells. Native gel electrophoresis showed that both p97 and p97(R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3 indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO/p97(R155C-RFP and ATG9(KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO mutant. A major finding is that the expression of p97(R155C-RFP in the ATG9(KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.

Expression of wild-type Rp1 protein in Rp1 knock-in mice rescues the retinal degeneration phenotype.

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Qin Liu

Full Text Available Mutations in the retinitis pigmentosa 1 (RP1 gene are a common cause of autosomal dominant retinitis pigmentosa (adRP, and have also been found to cause autosomal recessive RP (arRP in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39 are located in the 4(th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd exon of RP1 (c.686delC; p.P229QfsX35 found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.

White blood cells and subtypes in HFE p.C282Y and wild-type homozygotes in the Hemochromatosis and Iron Overload Screening Study.

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Barton, James C; Barton, J Clayborn; Acton, Ronald T

2017-03-01

The major histocompatibility complex is linked to white blood cell (WBC) and lymphocyte counts in subjects unselected for HFE genotypes. We compared age, sex, body mass index, total WBC and subtypes (neutrophils, lymphocytes, monocytes, eosinophils, basophils) (Beckman Coulter® Gen-S), transferrin saturation, and serum ferritin of HFE p.C282Y and wild-type (p.C282Y, p.H63D negative) homozygotes without acquired conditions that influence WBC counts. We performed regressions on WBC and subtypes. There were 161 p.C282Y homozygotes (45.3% men) and 221 wild-type homozygotes (40.3% men). Mean WBC of men and women and between HFE genotypes were similar. Mean lymphocytes were higher in male p.C282Y homozygotes: 1.6×10 9 /L [95% confidence interval: 1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002. Mean lymphocytes and basophils were higher in female p.C282Y homozygotes: 1.6 [1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002; and 0.065 [0.059,0.071] vs. 0.052 [0.051,0.054], p=0.0001, respectively. Transferrin saturation was associated with neutrophils (negative; p=0.0163). Age was associated with lymphocytes (negative; p=0.0003) and monocytes (positive; p<0.0001). Regressions on lymphocytes and basophils revealed positive associations with p.C282Y homozygosity (p=0.0043 and 0.0003, respectively). There were significant positive associations of neutrophils, lymphocytes, monocytes, and eosinophils. We conclude that HFE p.C282Y homozygosity is significantly associated with lymphocyte and basophil counts. Copyright © 2016 Elsevier Inc. All rights reserved.

Proline metabolism in the wild-type and in a salt-tolerant mutant of nicotiana plumbaginifolia studied by (13)C-nuclear magnetic resonance imaging

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Roosens; Willem; Li; Verbruggen; Biesemans; Jacobs

1999-12-01

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme.

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

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Zupan, Bojana; Toth, Miklos

2008-10-01

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

Anatomical location differences between mutated and wild-type isocitrate dehydrogenase 1 in low-grade gliomas.

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Yu, Jinhua; Shi, Zhifeng; Ji, Chunhong; Lian, Yuxi; Wang, Yuanyuan; Chen, Liang; Mao, Ying

2017-10-01

Anatomical location of gliomas has been considered as a factor implicating the contributions of a specific precursor cells during the tumor growth. Isocitrate dehydrogenase 1 (IDH1) is a pathognomonic biomarker with a significant impact on the development of gliomas and remarkable prognostic effect. The correlation between anatomical location of tumor and IDH1 states for low-grade gliomas was analyzed quantitatively in this study. Ninety-two patients diagnosed of low-grade glioma pathologically were recruited in this study, including 65 patients with IDH1-mutated glioma and 27 patients with wide-type IDH1. A convolutional neural network was designed to segment the tumor from three-dimensional magnetic resonance imaging images. Voxel-based lesion symptom mapping was then employed to study the tumor location distribution differences between gliomas with mutated and wild-type IDH1. In order to characterize the location differences quantitatively, the Automated Anatomical Labeling Atlas was used to partition the standard brain atlas into 116 anatomical volumes of interests (AVOIs). The percentages of tumors with different IDH1 states in 116 AVOIs were calculated and compared. Support vector machine and AdaBoost algorithms were used to estimate the IDH1 status based on the 116 location features of each patient. Experimental results proved that the quantitative tumor location measurement could be a very important group of imaging features in biomarker estimation based on radiomics analysis of glioma.

genetic overexpression of NR2B subunit enhances social recognition memory for different strains and species.

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Jacobs, Stephanie A; Tsien, Joe Z

2012-01-01

The ability to learn and remember conspecifics is essential for the establishment and maintenance of social groups. Many animals, including humans, primates and rodents, depend on stable social relationships for survival. Social learning and social recognition have become emerging areas of interest for neuroscientists but are still not well understood. It has been established that several hormones play a role in the modulation of social recognition including estrogen, oxytocin and arginine vasopression. Relatively few studies have investigated how social recognition might be improved or enhanced. In this study, we investigate the role of the NMDA receptor in social recognition memory, specifically the consequences of altering the ratio of the NR2B:NR2A subunits in the forebrain regions in social behavior. We produced transgenic mice in which the NR2B subunit of the NMDA receptor was overexpressed postnatally in the excitatory neurons of the forebrain areas including the cortex, amygdala and hippocampus. We investigated the ability of both our transgenic animals and their wild-type littermate to learn and remember juvenile conspecifics using both 1-hr and 24-hr memory tests. Our experiments show that the wild-type animals and NR2B transgenic mice preformed similarly in the 1-hr test. However, transgenic mice showed better performances in 24-hr tests of recognizing animals of a different strain or animals of a different species. We conclude that NR2B overexpression in the forebrain enhances social recognition memory for different strains and animal species.

Cognitive deficits in a genetic mouse model of the most common biochemical cause of human mental retardation.

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Zagreda, L; Goodman, J; Druin, D P; McDonald, D; Diamond, A

1999-07-15

Phenylalanine hydroxylase (Pah)-deficient "PKU mice" have a mutation in the Pah gene that causes phenylketonuria (PKU) in humans. PKU produces cognitive deficits in humans if it is untreated. We report here the first evidence that the genetic mouse model of PKU (Pah(enu2)) also produces cognitive impairments. PKU mice were impaired on both odor discrimination reversal and latent learning compared with heterozygote littermates and with wild-type mice of the same BTBR strain. A small container of cinnamon-scented sand was presented on the right or left, and nutmeg-scented sand was presented on the other side; left-right location varied over trials. Digging in sand of the correct scent was rewarded by finding phenylalanine-free chocolate. To prevent scent cuing, new containers were used on every trial, and both containers always contained chocolate. Digging in the incorrect choice was stopped before the chocolate was uncovered. Once criterion was reached, the other scent was rewarded. PKU mice were impaired on reversals 2, 3, and 4. They were also impaired in latent learning. On day 1, half the mice were allowed to explore a maze and discover the location of water. On day 2, all mice were water-deprived and were placed in the maze. Whereas pre-exposed wild-type and heterozygous mice showed evidence that they remembered the location of the water and hence could find the water faster on day 2, pre-exposed PKU mice showed no significant benefit from their pre-exposure on day 1.

Activity and Safety of Cetuximab Plus Modified FOLFOXIRI Followed by Maintenance With Cetuximab or Bevacizumab for RAS and BRAF Wild-type Metastatic Colorectal Cancer: A Randomized Phase 2 Clinical Trial.

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Cremolini, Chiara; Antoniotti, Carlotta; Lonardi, Sara; Aprile, Giuseppe; Bergamo, Francesca; Masi, Gianluca; Grande, Roberta; Tonini, Giuseppe; Mescoli, Claudia; Cardellino, Giovanni Gerardo; Coltelli, Luigi; Salvatore, Lisa; Corsi, Domenico Cristiano; Lupi, Cristiana; Gemma, Donatello; Ronzoni, Monica; Dell'Aquila, Emanuela; Marmorino, Federica; Di Fabio, Francesca; Mancini, Maria Laura; Marcucci, Lorenzo; Fontanini, Gabriella; Zagonel, Vittorina; Boni, Luca; Falcone, Alfredo

2018-04-01

The combination of a triple-drug chemotherapy regimen with an anti-epidermal growth factor receptor (EGFR) agent as a first-line treatment of metastatic colorectal cancer (mCRC) showed promising activity along with safety concerns in single-arm phase 2 trials. The role of maintenance following chemotherapy and anti-EGFR and the optimal regimen to be adopted are not established. To evaluate the activity and safety of cetuximab plus modified FOLFOXIRI (mFOLFOXIRI) and explore the role of maintenance with cetuximab or bevacizumab in RAS and BRAF wild-type mCRC. In a prospective, noncomparative, open-label, multicenter, randomized phase 2 trial, patients aged 18 to 75 years with unresectable, previously untreated RAS and BRAF wild-type (before amendment, KRAS wild-type) mCRC were recruited from 21 oncology units in Italy from October 19, 2011, to March 1, 2015 (followed up through May 31, 2017). In total, 323 patients were screened and 143 were randomized to 2 treatment arms to receive as a first-line induction a regimen of mFOLFOXIRI plus cetuximab followed by cetuximab (arm A) or bevacizumab (arm B) until disease progression. Primary analyses were conducted in a modified intention-to-treat population. mFOLFOXIRI plus cetuximab repeated every 2 weeks for up to 8 cycles, followed by maintenance with cetuximab or bevacizumab until disease progression. The primary end point was the 10-month progression-free rate (PFR); secondary end points included progression-free and overall survival, response rate, rate of metastases resection, and adverse events. Of 143 patients randomized, 116 (81.1%) (median [interquartile range (IQR)] age, 59.5 [53-67] years; 34 [29.3%] women) had RAS and BRAF wild-type mCRC. At a median (IQR) follow-up of 44.0 (30.5-52.1) months, 10-month PFRs were 50.8% (90% CI, 39.5%-62.2%) in arm A and 40.4% (90% CI, 29.4%-52.1%) in arm B. The overall response rate was 71.6% (95% CI, 62.4%-79.5%). Main grade 3/4 adverse events were neutropenia (occurring in 36

Primary tumor site is a useful predictor of cetuximab efficacy in the third-line or salvage treatment of KRAS wild-type (exon 2 non-mutant) metastatic colorectal cancer: a nationwide cohort study

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Chen, Kuo-Hsing; Shao, Yu-Yun; Chen, Ho-Min; Lin, Yu-Lin; Lin, Zhong-Zhe; Lai, Mei-Shu; Cheng, Ann-Lii; Yeh, Kun-Huei

2016-01-01

Previous studies have shown left-sided colorectal cancer (LCRC) and right-sided colorectal cancer (RCRC) exhibit different molecular and clinicopathological features. We explored the association between the primary tumor site and cetuximab efficacy in KRAS wild-type colorectal cancer (CRC). This study enrolled a cohort of patients, who had received cetuximab treatment after two or more lines of chemotherapy for KRAS wild-type (exon 2 nonmutant) metastatic CRC, from the databases of Taiwan Cancer Registry (2004–2010) and National Health Insurance (2004–2011). Survival data were obtained from the National Death Registry. Time to treatment discontinuation (TTD) and overall survival (OS) after the start of cetuximab treatment were compared between patients with LCRC (splenic flexure to rectum) and RCRC (cecum to hepatic flexure). A total of 969 CRC patients were enrolled. Among them, 765 (78.9 %) and 136 (14.0 %) patients had LCRC and RCRC, respectively. Patients with LCRC, compared to patients with RCRC, had longer TTD (median, 4.59 vs. 2.75 months, P = .0005) and OS (median, 12.62 vs. 8.07 months, P < .0001) after the start of cetuximab treatment. Multivariate analysis revealed a right-sided primary tumor site was an independent predictor of shorter TTD (adjusted hazard ratio [HR] = 1.32, using the LCRC group as a reference, 95 % confidence interval: 1.08–1.61, P = .0072) and OS (adjusted HR = 1.45, 95 % CI: 1.18–1.78, P = .0003). Our findings demonstrate that a left-sided primary tumor site is a useful predictor of improved cetuximab efficacy in the third-line or salvage treatment of KRAS wild-type (exon 2 nonmutant) metastatic CRC

Time Courses of Cortical Glucose Metabolism and Microglial Activity Across the Life Span of Wild-Type Mice: A PET Study.

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Brendel, Matthias; Focke, Carola; Blume, Tanja; Peters, Finn; Deussing, Maximilian; Probst, Federico; Jaworska, Anna; Overhoff, Felix; Albert, Nathalie; Lindner, Simon; von Ungern-Sternberg, Barbara; Bartenstein, Peter; Haass, Christian; Kleinberger, Gernot; Herms, Jochen; Rominger, Axel

2017-12-01

Contrary to findings in the human brain, 18 F-FDG PET shows cerebral hypermetabolism of aged wild-type (WT) mice relative to younger animals, supposedly due to microglial activation. Therefore, we used dual-tracer small-animal PET to examine directly the link between neuroinflammation and hypermetabolism in aged mice. Methods: WT mice (5-20 mo) were investigated in a cross-sectional design using 18 F-FDG ( n = 43) and translocator protein (TSPO) ( 18 F-GE180; n = 58) small-animal PET, with volume-of-interest and voxelwise analyses. Biochemical analysis of plasma cytokine levels and immunohistochemical confirmation of microglial activity were also performed. Results: Age-dependent cortical hypermetabolism in WT mice relative to young animals aged 5 mo peaked at 14.5 mo (+16%, P mice. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis.

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Scartozzi, Mario; Bearzi, Italo; Mandolesi, Alessandra; Pierantoni, Chiara; Loupakis, Fotios; Zaniboni, Alberto; Negri, Francesca; Quadri, Antonello; Zorzi, Fausto; Galizia, Eva; Berardi, Rossana; Biscotti, Tommasina; Labianca, Roberto; Masi, Gianluca; Falcone, Alfredo; Cascinu, Stefano

2009-08-27

K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN >or= 2.6 and CISH EGFR GCN >or= 2.12 and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

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Maria Fiorentino

Full Text Available Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa and might contribute (along with enterotoxins to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them

Canalization and control in automata networks: body segmentation in Drosophila melanogaster.

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Manuel Marques-Pita

Full Text Available We present schema redescription as a methodology to characterize canalization in automata networks used to model biochemical regulation and signalling. In our formulation, canalization becomes synonymous with redundancy present in the logic of automata. This results in straightforward measures to quantify canalization in an automaton (micro-level, which is in turn integrated into a highly scalable framework to characterize the collective dynamics of large-scale automata networks (macro-level. This way, our approach provides a method to link micro- to macro-level dynamics--a crux of complexity. Several new results ensue from this methodology: uncovering of dynamical modularity (modules in the dynamics rather than in the structure of networks, identification of minimal conditions and critical nodes to control the convergence to attractors, simulation of dynamical behaviour from incomplete information about initial conditions, and measures of macro-level canalization and robustness to perturbations. We exemplify our methodology with a well-known model of the intra- and inter cellular genetic regulation of body segmentation in Drosophila melanogaster. We use this model to show that our analysis does not contradict any previous findings. But we also obtain new knowledge about its behaviour: a better understanding of the size of its wild-type attractor basin (larger than previously thought, the identification of novel minimal conditions and critical nodes that control wild-type behaviour, and the resilience of these to stochastic interventions. Our methodology is applicable to any complex network that can be modelled using automata, but we focus on biochemical regulation and signalling, towards a better understanding of the (decentralized control that orchestrates cellular activity--with the ultimate goal of explaining how do cells and tissues 'compute'.

Canalization and control in automata networks: body segmentation in Drosophila melanogaster.

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Marques-Pita, Manuel; Rocha, Luis M

2013-01-01

We present schema redescription as a methodology to characterize canalization in automata networks used to model biochemical regulation and signalling. In our formulation, canalization becomes synonymous with redundancy present in the logic of automata. This results in straightforward measures to quantify canalization in an automaton (micro-level), which is in turn integrated into a highly scalable framework to characterize the collective dynamics of large-scale automata networks (macro-level). This way, our approach provides a method to link micro- to macro-level dynamics--a crux of complexity. Several new results ensue from this methodology: uncovering of dynamical modularity (modules in the dynamics rather than in the structure of networks), identification of minimal conditions and critical nodes to control the convergence to attractors, simulation of dynamical behaviour from incomplete information about initial conditions, and measures of macro-level canalization and robustness to perturbations. We exemplify our methodology with a well-known model of the intra- and inter cellular genetic regulation of body segmentation in Drosophila melanogaster. We use this model to show that our analysis does not contradict any previous findings. But we also obtain new knowledge about its behaviour: a better understanding of the size of its wild-type attractor basin (larger than previously thought), the identification of novel minimal conditions and critical nodes that control wild-type behaviour, and the resilience of these to stochastic interventions. Our methodology is applicable to any complex network that can be modelled using automata, but we focus on biochemical regulation and signalling, towards a better understanding of the (decentralized) control that orchestrates cellular activity--with the ultimate goal of explaining how do cells and tissues 'compute'.

Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1

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Murata, Kaoru; Hattori, Masakazu; Hirai, Norihito; Shinozuka, Yoriko; Hirata, Hiromi; Kageyama, Ryoichiro; Sakai, Toshiyuki; Minato, Nagahiro

2005-01-01

A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1. PMID:15870295

Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

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Shenghua Li

2009-08-01

Full Text Available The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella.

Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

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Li, Shenghua; Brazhnik, Paul; Sobral, Bruno; Tyson, John J

2009-08-01

The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella).

Retention of the In Vitro Radiosensitizing Potential of Gemcitabine Under Anoxic Conditions, in p53 Wild-Type and p53-Deficient Non-Small-Cell Lung Carcinoma Cells

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Wouters, An; Pauwels, Bea; Lambrechts, Hilde A.J.; Pattyn, Greet G.O.; Ides, Johan; Baay, Marc; Meijnders, Paul; Peeters, Marc; Vermorken, Jan B.; Lardon, Filip

2011-01-01

Purpose: Whereas radiosensitization by gemcitabine is well studied under normal oxygen conditions, little is known about its radiosensitizing potential under reduced oxygen conditions. Therefore, the present study evaluated the impact of anoxia on gemcitabine-mediated radiosensitization. Methods and Materials: The clonogenic assay was performed in three isogenic A549 cell lines differing in p53 status (24 h, 0-15 nM gemcitabine, 0-8 Gy irradiation, normoxia vs. anoxia). Using radiosensitizing conditions, cells were collected for cell cycle analysis and apoptosis detection. Results: Whereas wild-type p53 A549-LXSN cells were more sensitive to radiation than p53-deficient A549-E6 cells, both cell lines showed similar radiosensitization by gemcitabine under normoxia and anoxia. Independent of p53 functionality, gemcitabine was able to overcome anoxia-induced G 0/1 arrest and established an (early) S phase block in normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased with the gemcitabine/radiation combination, with a significant difference between A549-LXSN and A549-E6. Conclusions: This study is the first to show that gemcitabine retains its radiosensitizing potential under low oxygen conditions. Although radiosensitization was observed in both p53 wild-type and p53-deficient cells, p53 status might influence induction of apoptosis after gemcitabine/radiation treatment, whereas no effect on cell cycle progression was noticed.

Scanning Ultrasound (SUS Causes No Changes to Neuronal Excitability and Prevents Age-Related Reductions in Hippocampal CA1 Dendritic Structure in Wild-Type Mice.

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Robert John Hatch

Full Text Available Scanning ultrasound (SUS is a noninvasive approach that has recently been shown to ameliorate histopathological changes and restore memory functions in an Alzheimer's disease mouse model. Although no overt neuronal damage was reported, the short- and long-term effects of SUS on neuronal excitability and dendritic tree morphology had not been investigated. To address this, we performed patch-clamp recordings from hippocampal CA1 pyramidal neurons in wild-type mice 2 and 24 hours after a single SUS treatment, and one week and 3 months after six weekly SUS treatments, including sham treatments as controls. In both treatment regimes, no changes in CA1 neuronal excitability were observed in SUS-treated neurons when compared to sham-treated neurons at any time-point. For the multiple treatment groups, we also determined the dendritic morphology and spine densities of the neurons from which we had recorded. The apical trees of sham-treated neurons were reduced at the 3 month time-point when compared to one week; however, surprisingly, no longitudinal change was detected in the apical dendritic trees of SUS-treated neurons. In contrast, the length and complexity of the basal dendritic trees were not affected by SUS treatment at either time-point. The apical dendritic spine densities were reduced, independent of the treatment group, at 3 months compared to one week. Collectively, these data suggest that ultrasound can be employed to prevent an age-associated loss of dendritic structure without impairing neuronal excitability.

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

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Ramoni, Jonas; Marchetti-Deschmann, Martina; Seidl-Seiboth, Verena; Seiboth, Bernhard

2017-05-01

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

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Saara Laulumaa

Full Text Available Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS. The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

Proline Metabolism in the Wild-Type and in a Salt-Tolerant Mutant of Nicotiana plumbaginifolia Studied by 13C-Nuclear Magnetic Resonance Imaging1

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Roosens, Nancy H.; Willem, Rudolph; Li, Yan; Verbruggen, Ingrid; Biesemans, Monique; Jacobs, Michel

1999-01-01

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. 13C-Nuclear magnetic resonance with [5-13C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mm NaCl stress in the presence of [5-13C]Glu, a significant enrichment in [5-13C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-13C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-13C]Glu into [5-13C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Δ-pyrroline-5-carboxylate synthetase enzyme. PMID:10594115

Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci.

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Schulte, B A; Steel, K P

1994-07-01

Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final

Late inflammatory and thrombotic changes in irradiated hearts of C57BL/6 wild-type and atherosclerosis-prone ApoE-deficient mice

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Patties, I.; Glasow, A. [University of Leipzig, Department of Radiation Therapy, Leipzig (Germany); Haagen, J. [University of Technology, Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Dresden (Germany); Doerr, W. [University of Technology, Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Dresden (Germany); CCC, Medical University/AKH, Department of Radiation Oncology and Christian Doppler Laboratory for Medical Radiation Research for Radiooncology, Vienna (Austria); Hildebrandt, G. [University of Rostock, Department of Radiotherapy and Radiation Oncology, Rostock (Germany)

2014-09-09

Radiation-induced heart disease represents a late complication of thoracic radiotherapy. We investigated the inflammatory and thrombotic response after local heart irradiation in wild-type and atherosclerosis-prone mice. Atherosclerosis-prone ApoE{sup -/-} and C57BL/6 wild-type mice were sacrificed 20, 40, and 60 weeks after irradiation with 0.2, 2, 8, or 16 Gy. The expression of CD31, vascular cell adhesion molecule-1 (VCAM-1), thrombomodulin (TM), and CD45 were quantified by immunofluorescence staining of heart tissue sections. Microvascular density decreased at 40 weeks after 16 Gy in C57BL/6 but not in ApoE{sup -/-} mice. CD31 expression declined in C57BL/6 mice at 40 weeks (8 Gy), but increased in ApoE{sup -/-} mice at 20 (2/8/16 Gy) and 60 weeks (16 Gy). Capillary area decreased in C57BL/6 at 40 weeks (8/16 Gy) but increased in ApoE{sup -/-} mice at 20 weeks (16 Gy). Endocardial VCAM-1 expression remained unchanged. TM-positive capillaries decreased at 40 weeks (8/16 Gy) in C57BL/6 and at 60 weeks (2/16 Gy) in ApoE{sup -/-} mice. Leukocyte infiltration transiently rose 40 weeks after 8 Gy (only ApoE{sup -/-}) and 16 Gy. After receiving a low irradiation dose of 0.2 Gy, no significant changes were observed in any of the mouse models. This study demonstrated that local heart irradiation affects microvascular structure and induces inflammatory/thrombotic responses in mice in a dose- and time-dependent manner. Thereby, significant prothrombotic changes were found in both strains, although they were progressive in ApoE{sup -/-} mice only. Proinflammatory responses, like the increase of adhesion molecules and leukocyte infiltration, were more pronounced and occurred at lower doses in ApoE{sup -/-} vs. C57BL/6 mice. These findings indicate that metabolic risk factors, such as decreased ApoE lipoproteins, may lead to an enhanced proinflammatory and prothrombotic late response in locally irradiated hearts. (orig.) [German] Strahlungsinduzierte kardiovaskulaere

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection.

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Andreas Grahnert

Full Text Available Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα⁻/⁻ mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα⁻/⁻ mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα⁻/⁻ mice compared to wild-type mice. To directly study the potential mechanism(s responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar

DEFF Research Database (Denmark)

Dubois, A. E. J.; Pagliarani, G.; Brouwer, R. M.

2015-01-01

BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples...... which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual...

Maid (GCIP) is involved in cell cycle control of hepatocytes

DEFF Research Database (Denmark)

Sonnenberg-Riethmacher, Eva; Wüstefeld, Torsten; Miehe, Michaela

2007-01-01

. Therefore, we studied the role of Maid during cell cycle progression after partial hepatectomy (PH). Lack of Maid expression after PH was associated with a delay in G1/S-phase progression as evidenced by delayed cyclinA expression and DNA replication in Maid-deficient mice. However, at later time points......The function of Maid (GCIP), a cyclinD-binding helix-loop-helix protein, was analyzed by targeted disruption in mice. We show that Maid function is not required for normal embryonic development. However, older Maid-deficient mice-in contrast to wild-type controls--develop hepatocellular carcinomas...

Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

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Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G

2001-01-01

VMAT2 expression in wild-type and DAT knockout midbrain. DAT immunoreactivity in wild-type (A) and DAT knockout midbrain (B). VMAT2 immunoreactivity in wild-type (C) and DAT knockout midbrain (D). Robust immunoreactivity was observed in the ventral tegmental area and substantia nigra pars compacta and reticulata in the wild-type brain. Note absence of DAT immunoreactivity and modest reduction of VMAT2 immunoreactivity in the DAT knockout. Fig. 3. Characterization of VMAT2 gene disruption. (A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4-kb 3' external genomic fragment. +/+, wild type littermates; +/-, heterozygote; -/-, homozygote. (B) RT-PCR analysis of mouse brain poly(A)+ RNA. For each reverse transcription assay, 0.5 μg of poly(A)+ RNA was used. Equal volumes of cDNA templates were used for each PCR assay. The PCR primers used flank the neomycin cassette for the purpose of detecting potential readthrough of the neomycin DNA. The heterozygote has a reduced amount of transcripts compared with the wild-type littermate; the homozygote is devoid of VMAT2 transcripts. G3PDH was used as internal control. (C) Western blot analysis of wholebrain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by SDS-PAGE, transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti-a-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown to the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. (Reproduced with permission from ref. 1). The importance of DAT in neuronal function is highlighted in animals in which DAT has been genetically deleted (DAT KO) (3). In the homozygote DAT KO mice, released dopamine remains in the extracellular space up to 300 times longer than normal. As expected, these animals display

Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice

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Fernanda S. Oliveira

2012-01-01

Full Text Available Nucleotide-binding oligomerization domain proteins (NODs are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.

Wild-type EGFR Is Stabilized by Direct Interaction with HSP90 in Cancer Cells and Tumors

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Aarif Ahsan

2012-08-01

Full Text Available The epidermal growth factor receptor (EGFR has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90 is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function.

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Zhang, Yu; Patel, Bella; Dey, Aditi; Ghorani, Ehsan; Rai, Lena; Elham, Mohammed; Castleton, Anna Z; Fielding, Adele K

2012-02-01

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

Development, anatomy, and genetic control of some teratological phenotypes of Ranunculaceae flowers

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Florian Jabbour

2016-04-01

Full Text Available Teratological organisms originate from developmental anomalies, and exhibit structures and a body organization that deviate from the species standard. These monsters give essential clues about the formation and evolutionary significance of the wild-type groundplan. We focus on flower terata, which can be affected in their sterile and/or fertile organs, with special emphasis on the Ranunculaceae. The diversity of perianth shapes and organizations in flowers of this family is huge, and is even increased when anomalies occur during organo- and/or morphogenesis. To begin with, we synthesize the observations and research conducted on the Ranunculacean floral terata, following the most recent phylogenetic framework published in 2016 by our team. Then, we report results regarding the morphology of developing meristems, the anatomy of buds, and the genetic control of selected teratological phenotypes of Ranunculaceae flowers. We focus on species and horticultural varieties belonging to the genera Aquilegia, Delphinium, and Nigella. Wildtype flowers of these species are actinomorphic (Aquilegia, Nigella or zygomorphic (Delphinium, spurred (Aquilegia, Delphinium or with pocket-like petals (Nigella. Last, we discuss the evolutionary potential of such teratological phenotypes when they occur in the wild.

Differential Activity of the Oral Glucan Synthase Inhibitor SCY-078 against Wild-Type and Echinocandin-Resistant Strains of Candida Species.

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Pfaller, Michael A; Messer, Shawn A; Rhomberg, Paul R; Borroto-Esoda, Katyna; Castanheira, Mariana

2017-08-01

SCY-078 (formerly MK-3118) is a novel orally active inhibitor of fungal β-(1,3)-glucan synthase (GS). SCY-078 is a derivative of enfumafungin and is structurally distinct from the echinocandin class of antifungal agents. We evaluated the in vitro activity of this compound against wild-type (WT) and echinocandin-resistant isolates containing mutations in the FKS genes of Candida spp. Against 36 Candida spp. FKS mutants tested, 30 (83.3%) were non-WT to 1 or more echinocandins, and only 9 (25.0%) were non-WT (MIC, >WT-upper limit) to SCY-078. Among C. glabrata isolates carrying FKS alterations, 84.0% were non-WT to the echinocandins versus only 24.0% for SCY-078. In contrast to the echinocandin comparators, the activity of SCY-078 was minimally affected by the presence of FKS mutations, suggesting that this agent is useful in the treatment of Candida infections due to echinocandin-resistant strains. Copyright © 2017 American Society for Microbiology.

Swine dysentery: protection of pigs by oral and parenteral immunisation with attenuated Treponema hyodysenteriae.

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Hudson, M J; Alexander, T J; Lysons, R J; Prescott, J F

1976-11-01

An attenuated strain of Treponema hyodysenteriae was used to immunise 18 pigs in three experiments. Live attenuated spirochaetes were dosed orally and injected intra-peritoneally, and killed spirochaetes were injected intramuscularly with adjuvant. The vaccinated pigs, which developed high serum agglutination titres against T hyodysenteriae, and 18 unvaccinated litter-mates were repeatedly challenged with virulent T hyodysenteriae. Nine vaccinated pigs and 16 control pigs developed typical swine dysentery.

A Longitudinal Study of Cognition, Proton MR Spectroscopy and Synaptic and Neuronal Pathology in Aging Wild-type and AβPPswe-PS1dE9 Mice

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Jansen, Diane; Zerbi, Valerio; Janssen, Carola I. F.; Dederen, Pieter J. W. C.; Mutsaers, Martina P. C.; Hafkemeijer, Anne; Janssen, Anna-Lena; Nobelen, Cindy L. M.; Veltien, Andor; Asten, Jack J.; Heerschap, Arend; Kiliaan, Amanda J.

2013-01-01

Proton magnetic resonance spectroscopy (1H MRS) is a valuable tool in Alzheimer’s disease research, investigating the functional integrity of the brain. The present longitudinal study set out to characterize the neurochemical profile of the hippocampus, measured by single voxel 1H MRS at 7 Tesla, in the brains of AβPPSswe-PS1dE9 and wild-type mice at 8 and 12 months of age. Furthermore, we wanted to determine whether alterations in hippocampal metabolite levels coincided with behavioral changes, cognitive decline and neuropathological features, to gain a better understanding of the underlying neurodegenerative processes. Moreover, correlation analyses were performed in the 12-month-old AβPP-PS1 animals with the hippocampal amyloid-β deposition, TBS-T soluble Aβ levels and high-molecular weight Aβ aggregate levels to gain a better understanding of the possible involvement of Aβ in neurochemical and behavioral changes, cognitive decline and neuropathological features in AβPP-PS1 transgenic mice. Our results show that at 8 months of age AβPPswe-PS1dE9 mice display behavioral and cognitive changes compared to age-matched wild-type mice, as determined in the open field and the (reverse) Morris water maze. However, there were no variations in hippocampal metabolite levels at this age. AβPP-PS1 mice at 12 months of age display more severe behavioral and cognitive impairment, which coincided with alterations in hippocampal metabolite levels that suggest reduced neuronal integrity. Furthermore, correlation analyses suggest a possible role of Aβ in inflammatory processes, synaptic dysfunction and impaired neurogenesis. PMID:23717459

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study.

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Wang, Ruoqing; Zhang, Nianwei; Wang, Renkai; Wang, Shengpeng; Wang, Na

2017-12-01

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

Light induced apoptosis is accelerated in transgenic retina overexpressing human EAT/mcl-1, an anti-apoptotic bcl-2 related gene.

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Shinoda, K; Nakamura, Y; Matsushita, K; Shimoda, K; Okita, H; Fukuma, M; Yamada, T; Ohde, H; Oguchi, Y; Hata, J; Umezawa, A

2001-10-01

EAT/mcl-1 (EAT), an immediate early gene, functions in a similar way to bcl-2 in neutralising Bax mediated cytotoxicity, suggesting that EAT is a blocker of cell death. The aim of this study was to determine the effect of overexpression of the human EAT gene on light induced retinal cell apoptosis. EAT transgenic mice incorporating the EF-1alpha promoter were utilised, and expression of human EAT was detected by RT-PCR. Light damage was induced by raising mice under constant illumination. Two groups of animals, EAT transgenic mice (n=14) and littermates (n=13), were examined by ERG testing and histopathology at regular time points up to 20 weeks of constant light stimulation. Electrophysiological and histopathological findings were evaluated by established systems of arbitrary scoring as scores 0-2 and scores 0-3, respectively. The mean score (SD) of ERG response was significantly lower in EAT transgenic mice (0.79 (0.89)) than in littermates (1.69 (0.48)) (pstatistical significance (p=0.1156), the estimated incidence of electrophysiological retinal damage was higher in EAT mice (0.0495/mouse/week; 95% confidence interval (CI) 0.0347-0.0500) than in littermates (0. 0199/mouse/week; 95% CI 0.0035-0.0364). The mean scores (SD) for histopathological retinal degeneration were 2.31 (0.63) in littermates and 1.43 (1.22) in EAT transgenic mice (p=0.065). However, Kaplan-Meier curves for histopathological failure in two groups of mice showed that retinal photoreceptor cells were preserved significantly against constant light in the littermate compared with transgenic mice (p=0.0241). The estimated incidence of histopathological retinal damage was 0.0042/mouse/week in the littermates (95% CI 0-0.0120) and 0.0419/mouse/week in the EAT mice (95% CI 0.0286-0.0500). Retinal photoreceptor cell apoptosis under constant light stimulation is likely to be accelerated in transgenic retina overexpressing EAT.

Multilineage dysplasia is associated with a poorer prognosis in patients with de novo acute myeloid leukemia with intermediate-risk cytogenetics and wild-type NPM1.

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Rozman, María; Navarro, José-Tomás; Arenillas, Leonor; Aventín, Anna; Giménez, Teresa; Alonso, Esther; Perea, Granada; Camós, Mireia; Navarrete, Mayda; Tuset, Esperanza; Florensa, Lourdes; Millá, Fuensanta; Nomdedéu, Josep; de la Banda, Esmeralda; Díaz-Beyá, Marina; Pratcorona, Marta; Garrido, Ana; Navarro, Blanca; Brunet, Salut; Sierra, Jorge; Esteve, Jordi

2014-10-01

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

Evidence for a second 'Prereplicative G2' repair mechanism, specific for γ-induced damage, in wild-type schizosaccharomyces pombe

International Nuclear Information System (INIS)

Gentner, N.E.; Atomic Energy of Canada Ltd., Chalk River, Ontario. Chalk River Nuclear Labs.)

1977-01-01

The major part of the substantial γ-resistance of wild-type Schizosaccharomyces pombe appears to be due to prereplicative recombinational repair mechanisms. The existence of a second 'prereplicative G2' repair pathway, specific for γ-induced damage, has now been deduced from studies of the effect of the repair inhibitor caffeine on γ-irradiated G1 phase and G2 phase cells. Only G2 cells are additionally inactivated on exposure to caffeine after γ-irradiation. This shows that both known caffeine-sensitive γ-repair processes (Genter and Werner, Molec. gen. Genet. 145, 1-5 [1976]) are dependent on the presence of a duplicated genome (2c) at the time of radiation exposure. Pathway I is the known 'prereplicative G2' repair process (Fabre, Radiation Res. 56, 528-539 [1973]) which is involved in both UV- and γ-repair, and which requires post-irradiation protein synthesis for activity. Pathway II represents a second distinct 'prereplicative G2' repair mechanism; it differs from the first in that it is specific for repair of γ-induced damage and appears to be constitutive. (orig.) [de

EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin.

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Van Doorslaer, Sabine; Trandafir, Florin; Harmer, Jeffrey R; Moens, Luc; Dewilde, Sylvia

2014-06-01

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric ((13)C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full (13)C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on (13)C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding (13)C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These (13)C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Production and purification of anti-bacterial biometabolite from wild-type Lactobacillus, isolated from fermented bamboo shoot: future suggestions and a proposed system for secondary metabolite onsite recovery during continuous fermentation.

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Badwaik, Laxmikant S; Borah, Pallab Kumar; Deka, Sankar C

2015-02-01

Wild-type lactobacillus isolated form Khorisa, a fermented bamboo shoot product of Assam, India were evaluated for production anti-bacterial secondary biometabolites, against Staphylococcus aureus. Submerged fermentation technique was used for the production of secondary anti-microbial biometabolite by a single wild-type lactobacillus strain, which tested positive for the release of anti-bacterial factor(s). Crude cell-free supernatant was obtained, followed by extraction in water-immiscible solvents viz., chloroform, hexane, petroleum ether. Chloroform extract of cell-free crude supernatant showed maximum yield (0.054 g/ml) and inhibited all indicator bacterial strains viz., Escherichia coli, Staphylococcus aureus, and Bacillus cereus. Yields of hexane and petroleum ether extract were 0.052 and 0.026 g/ml, respectively. Minimum lethal dose concentration assay of the chloroform extract showed LDmin values at 27, 1.68, and 1.68 mg/ml for E. coli, S. aureus, and B. cereus, respectively. Kill time for all the indicator bacterial strains were less than 12 h. The efficacy of the anti-bacterial substance seemed to depend on the presence of organic acids, particularly lactic acid. Conceptual-based suggestion for the development of an onsite secondary metabolites recovery system during continuous fermentation has also been attempted.

Multipulse spectroscopy on the wild-type and YM210W Bacterial Reaction Centre uncovers a new intermediate state in the special pair excited state

Science.gov (United States)

Cohen Stuart, T. A.; van Grondelle, R.

2009-06-01

The Bacterial Reaction Centre (BRC) has a complex electronic excited state, P ∗, that evolves into subsequent charge separated product states P +H - and P +B -. Pump-dump-probe spectroscopy on the wild-type BRC and on YM210W, a mutant with a stabilized, long-lived P ∗ excited state, has uncovered a new charge-separated state in both BRC's. When P ∗ is dumped, a fraction of its population is transferred to this state that has a strong Stark shift in the accessory bacteriochlorophyll (B M) region which serves as a signature for P + and a lifetime highly comparable to the slow phase of P ∗ decay. This lead us propose this intermediate to be P +/P -.

Neurokinin B is critical for normal timing of sexual maturation but dispensable for adult reproductive function in female mice.

Science.gov (United States)

True, Cadence; Nasrin Alam, Sayeda; Cox, Kimberly; Chan, Yee-Ming; Seminara, Stephanie B

2015-04-01

Humans carrying mutations in neurokinin B (NKB) or the NKB receptor fail to undergo puberty due to decreased secretion of GnRH. Despite this pubertal delay, many of these patients go on to achieve activation of their hypothalamic-pituitary-gonadal axis in adulthood, a phenomenon termed reversal, indicating that NKB signaling may play a more critical role for the timing of pubertal development than adult reproductive function. NKB receptor-deficient mice are hypogonadotropic but have no defects in the timing of sexual maturation. The current study has performed the first phenotypic evaluation of mice bearing mutations in Tac2, the gene encoding the NKB ligand, to determine whether they have impaired sexual development similar to their human counterparts. Male Tac2-/- mice showed no difference in the timing of sexual maturation or fertility compared with wild-type littermates and were fertile. In contrast, Tac2-/- females had profound delays in sexual maturation, with time to vaginal opening and first estrus occurring significantly later than controls, and initial abnormalities in estrous cycles. However, cycling recovered in adulthood and Tac2-/- females were fertile, although they produced fewer pups per litter. Thus, female Tac2-/- mice parallel humans harboring NKB pathway mutations, with delayed sexual maturation and activation of the reproductive cascade later in life. Moreover, direct comparison of NKB ligand and receptor-deficient females confirmed that only NKB ligand-deficient animals have delayed sexual maturation, suggesting that in the absence of the NKB receptor, NKB may regulate the timing of sexual maturation through other tachykinin receptors.

Stereological Investigation of the Effects of Treadmill Running Exercise on the Hippocampal Neurons in Middle-Aged APP/PS1 Transgenic Mice.

Science.gov (United States)

Chao, Fenglei; Jiang, Lin; Zhang, Yi; Zhou, Chunni; Zhang, Lei; Tang, Jing; Liang, Xin; Qi, Yingqiang; Zhu, Yanqing; Ma, Jing; Tang, Yong

2018-01-01

The risk of cognitive decline during Alzheimer's disease (AD) can be reduced if physical activity is maintained; however, the specific neural events underlying this beneficial effect are still uncertain. To quantitatively investigate the neural events underlying the effect of running exercise on middle-aged AD subjects, 12-month-old male APP/PS1 mice were randomly assigned to a control group or running group, and age-matched non-transgenic littermates were used as a wild-type group. AD running group mice were subjected to a treadmill running protocol (regular and moderate intensity) for four months. Spatial learning and memory abilities were assessed using the Morris water maze. Hippocampal amyloid plaques were observed using Thioflavin S staining and immunohistochemistry. Hippocampal volume, number of neurons, and number of newborn cells (BrdU+ cells) in the hippocampus were estimated using stereological techniques, and newborn neurons were observed using double-labelling immunofluorescence. Marked neuronal loss in both the CA1 field and dentate gyrus (DG) and deficits in both the neurogenesis and survival of new neurons in the DG of middle-aged APP/PS1 mice were observed. Running exercise could improve the spatial learning and memory abilities, reduce amyloid plaques in the hippocampi, delay neuronal loss, induce neurogenesis, and promote the survival of newborn neurons in the DG of middle-aged APP/PS1 mice. Exercise-induced protection of neurons and adult neurogenesis within the DG might be part of the important structural basis of the improved spatial learning and memory abilities observed in AD mice.