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Sample records for mice reveals major

  1. Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

    PubMed

    Flodby, Per; Kim, Yong Ho; Beard, LaMonta L; Gao, Danping; Ji, Yanbin; Kage, Hidenori; Liebler, Janice M; Minoo, Parviz; Kim, Kwang-Jin; Borok, Zea; Crandall, Edward D

    2016-09-01

    Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

  2. Mice selected for extremes in stress reactivity reveal key endophenotypes of major depression: a translational approach.

    PubMed

    Heinzmann, Jan-Michael; Kloiber, Stefan; Ebling-Mattos, Gabriele; Bielohuby, Maximilian; Schmidt, Mathias V; Palme, Rupert; Holsboer, Florian; Uhr, Manfred; Ising, Marcus; Touma, Chadi

    2014-11-01

    Clear evidence has linked dysregulated hypothalamus-pituitary-adrenocortical (HPA) axis function to the aetiology and pathophysiology of major depression (MD), as observed in the majority of patients. Increased stress reactivity and hyperactivity of the HPA axis seem characteristic for psychotic/melancholic depression, while the atypical subtype of depression has been connected with the opposing phenotypes. However, the underlying molecular-genetic mechanisms are poorly understood. In the present study, mouse lines selectively bred for extremes in stress reactivity (SR), i.e. presenting high (HR) or low (LR) corticosterone secretion in response to stressors, were used to characterise the molecular alterations on all levels of the HPA axis. Results were contrasted with clinical phenotypes of MD patients from the Munich Antidepressant Response Signature project, stratified according to their cortisol response in the Dex/CRH test. Distinct differences between HR and LR mice were found in the expression of HPA axis-related genes in the adrenals, pituitary and selected brain areas. Moreover, HR animals presented an enhanced adrenal sensitivity, increased stress-induced neuronal activation in the PVN and an overshooting Dex/CRH test response, whereas LR animals showed a blunted response in these paradigms. Interestingly, analogous neuroendocrine, morphometric, psychopathological and behavioural differences were observed between the respective high and low HPA axis responder groups of MD patients. Our findings suggests that (i) the SR mouse model can serve as a valuable tool to elucidate HPA axis-related mechanisms underlying affective disorders and (ii) a stratification of MD patients according to their HPA axis-related neuroendocrine function should be considered for clinical research and treatment.

  3. Inbred Strains Derived from Feral Mice Reveal New Pathogenic Mechanisms of Experimental Leishmaniasis Due to Leishmania major

    PubMed Central

    Babay, Besma E. C.; Louzir, Hechmi; Kebaïer, Chahnaz; Boubaker, Samir; Dellagi, Koussay; Cazenave, Pierre-André

    2004-01-01

    Two inbred mouse strains, derived from feral founders, are susceptible to experimental leishmaniasis due to Leishmania major and support a disease of a severity intermediate between those observed in strains C57BL/6 and BALB/c. Mice of the MAI strain develop a severe, nonhealing, but nonfatal disease with no resistance to a secondary parasite challenge. The immunological responses showed a TH2 dominance characterized by an early peak of interleukin-4 (IL-4) and IL-13. However, neutralization of IL-4, which leads to a resistance phenotype in BALB/c mice, has no effect on disease progression in MAI mice. Mice of strain PWK develop a protracted but self-healing disease, characterized by a mixed TH1-plus-TH2 pattern of immune responses in which IL-10 plays an aggravating role, and acquire resistance to a secondary challenge. These features are close to those observed in human cutaneous leishmaniasis due to L. major and make PWK mice a suitable model for the human disease. PMID:15271920

  4. Inbred strains derived from feral mice reveal new pathogenic mechanisms of experimental leishmaniasis due to Leishmania major.

    PubMed

    Babay, Besma E C; Louzir, Hechmi; Kebaïer, Chahnaz; Boubaker, Samir; Dellagi, Koussay; Cazenave, Pierre-André

    2004-08-01

    Two inbred mouse strains, derived from feral founders, are susceptible to experimental leishmaniasis due to Leishmania major and support a disease of a severity intermediate between those observed in strains C57BL/6 and BALB/c. Mice of the MAI strain develop a severe, nonhealing, but nonfatal disease with no resistance to a secondary parasite challenge. The immunological responses showed a TH2 dominance characterized by an early peak of interleukin-4 (IL-4) and IL-13. However, neutralization of IL-4, which leads to a resistance phenotype in BALB/c mice, has no effect on disease progression in MAI mice. Mice of strain PWK develop a protracted but self-healing disease, characterized by a mixed TH1-plus-TH2 pattern of immune responses in which IL-10 plays an aggravating role, and acquire resistance to a secondary challenge. These features are close to those observed in human cutaneous leishmaniasis due to L. major and make PWK mice a suitable model for the human disease.

  5. Proteomic Profiling of Adipose Tissue from Zmpste24−/− Mice, a Model of Lipodystrophy and Premature Aging, Reveals Major Changes in Mitochondrial Function and Vimentin Processing*

    PubMed Central

    Peinado, Juan R.; Quirós, Pedro M.; Pulido, Marina R.; Mariño, Guillermo; Martínez-Chantar, Maria L.; Vázquez-Martínez, Rafael; Freije, José M. P.; López-Otín, Carlos; Malagón, María M.

    2011-01-01

    Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24−/− mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24−/− mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24−/− serum. Together, our findings in Zmpste24−/− mice may be useful to unveil the mechanisms underlying adipose

  6. Connexin43 null mice reveal that astrocytes express multiple connexins.

    PubMed

    Dermietzel, R; Gao, Y; Scemes, E; Vieira, D; Urban, M; Kremer, M; Bennett, M V; Spray, D C

    2000-04-01

    The gap junction protein connexin43 (Cx43) is the primary component of intercellular channels in cardiac tissue and in astrocytes, the most abundant type of glial cells in the brain. Mice in which the gene for Cx43 is deleted by homologous recombination die at birth, due to profound hypertrophy of the ventricular outflow tract and stenosis of the pulmonary artery. Despite this significant cardiovascular abnormality, brains of connexin43 null [Cx43 (-/-)] animals are shown to be macroscopically normal and to display a pattern of cortical lamination that is not detectably different from wildtype siblings. Presence of Cx40 and Cx45 in brains and astrocytes cultured from both Cx43 (-/-) mice and wildtype littermates was confirmed by RT-PCR, Northern blot analyses and by immunostaining; Cx46 was detected by RT-PCR and Northern blot analyses. Presence of Cx26 in astrocyte cultures was indicated by RT-PCR and by Western blot analysis, although we were unable to resolve whether it was contributed by contaminating cells; Cx30 mRNA was detected by Northern blot in long term (2 weeks) but not fresh cultures of astrocytes. These studies thus reveal that astrocyte gap junctions may be formed of multiple connexins. Presumably, the metabolic and ionic coupling provided by these diverse gap junction types may functionally compensate for the absence of the major astrocyte gap junction protein in Cx43 (-/-) mice, providing whatever intercellular signaling is necessary for brain development and cortical lamination.

  7. Major contribution of tubular secretion to creatinine clearance in mice

    PubMed Central

    Eisner, Christoph; Faulhaber-Walter, Robert; Wang, Yaohui; Leelahavanichkul, Asada; Yuen, Peter S.T.; Mizel, Diane; Star, Robert A.; Briggs, Josephine P.; Levine, Mark; Schnermann, Jurgen

    2011-01-01

    This study was performed to quantify the fraction of excreted creatinine not attributable to creatinine filtration for accurately determining the glomerular filtration rate in mice. To measure this we compared creatinine filtration with the simultaneous measurement of inulin clearance using both single-bolus fluorescein isothiocyanate (FITC)-inulin elimination kinetics and standard FITC-inulin infusion. During anesthesia, creatinine filtration was found to be systematically higher than inulin clearance in both male and female C57BL/6J mice. The secretion fraction was significantly less in female mice. Administration of either cimetidine or para-aminohippuric acid, competitors of organic cation and anion transport respectively, significantly reduced the secretion fraction in male and female mice and both significantly increased the plasma creatinine level. Creatinine secretion in both genders was not mediated by the organic cation transporters OCT1 or OCT 2 since secretion fraction levels were identical in FVB wild-type and OCT1/2 knockout mice. Thus, secretion accounts for about 50 and 35% of excreted creatinine in male and female mice, respectively. Increasing plasma creatinine threefold by infusion further increased the secretion fraction. Renal organic anion transporter 1 mRNA expression was higher in male than in female mice, reflecting the gender difference in creatinine secretion. Hence we show that there is a major secretory contribution to creatinine excretion mediated through the organic anion transport system. This feature adds to problems associated with measuring endogenous creatinine filtration in mice. PMID:20032962

  8. Effect of Amphotericin B Nanodisks on Leishmania major Infected Mice

    PubMed Central

    Cole, PA; Bishop, JV; Beckstead, JA; Titus, R; Ryan, RO

    2014-01-01

    Objective To assess the efficacy of a novel formulation of the polyene antibiotic, amphotericin B (AMB), as therapy for cutaneous leishmaniasis in different mouse strains. Methods (AMB), was formulated into water-soluble transport particles, termed nanodisks (ND). Balb/c and CH3 mice infected with Leishmania major on Day 0 were administered vehicle alone, empty ND or AMB-ND on Day 1 and day 7, via the tail vein. Mice were sacrificed 25 or 50 days post inoculation and tissue histology evaluated. Balb/c mice treated with vehicle or empty ND showed signs of severe infection while CH3 mice had less inflammation and fewer parasites. AMB-ND treatment (2 mg/kg) had a marked therapeutic effect on L. major infected Balb/c mice and a discernable therapeutic benefit on CH3 mice. Conclusions AMB-ND is efficacious in the treatment of cutaneous leishmaniasis in both susceptible and resistant mouse strains. It may be inferred that AMB-ND may be useful for prophylactic and/or treatment of early stage Leishmania spp. infection. PMID:25584195

  9. Mice completely lacking immunoproteasomes display major alterations in antigen presentation

    PubMed Central

    Kincaid, Eleanor Z; Che, Jenny W; York, Ian; Escobar, Hernando; Reyes-Vargas, Eduardo; Delgado, Julio C.; Welsh, Raymond M; Karow, Margaret L.; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rock, Kenneth L

    2011-01-01

    The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought. PMID:22197977

  10. Diversity of major urinary proteins (MUPs) in wild house mice

    PubMed Central

    Thoß, Michaela; Enk, Viktoria; Yu, Hans; Miller, Ingrid; Luzynski, Kenneth C.; Balint, Boglarka; Smith, Steve; Razzazi-Fazeli, Ebrahim; Penn, Dustin J.

    2016-01-01

    Major urinary proteins (MUPs) are often suggested to be highly polymorphic, and thereby provide unique chemical signatures used for individual and genetic kin recognition; however, studies on MUP variability have been lacking. We surveyed populations of wild house mice (Mus musculus musculus), and examined variation of MUP genes and proteins. We sequenced several Mup genes (9 to 11 loci) and unexpectedly found no inter-individual variation. We also found that microsatellite markers inside the MUP cluster show remarkably low levels of allelic diversity, and significantly lower than the diversity of markers flanking the cluster or other markers in the genome. We found low individual variation in the number and types of MUP proteins using a shotgun proteomic approach, even among mice with variable MUP electrophoretic profiles. We identified gel bands and spots using high-resolution mass spectrometry and discovered that gel-based methods do not separate MUP proteins, and therefore do not provide measures of MUP diversity, as generally assumed. The low diversity and high homology of Mup genes are likely maintained by purifying selection and gene conversion, and our results indicate that the type of selection on MUPs and their adaptive functions need to be re-evaluated. PMID:27922085

  11. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.

    PubMed

    Cho, Joo-Youn; Matsubara, Tsutomu; Kang, Dong Wook; Ahn, Sung-Hoon; Krausz, Kristopher W; Idle, Jeffrey R; Luecke, Hans; Gonzalez, Frank J

    2010-05-01

    Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

  12. Seafood prices reveal impacts of a major ecological disturbance.

    PubMed

    Smith, Martin D; Oglend, Atle; Kirkpatrick, A Justin; Asche, Frank; Bennear, Lori S; Craig, J Kevin; Nance, James M

    2017-02-14

    Coastal hypoxia (dissolved oxygen ≤ 2 mg/L) is a growing problem worldwide that threatens marine ecosystem services, but little is known about economic effects on fisheries. Here, we provide evidence that hypoxia causes economic impacts on a major fishery. Ecological studies of hypoxia and marine fauna suggest multiple mechanisms through which hypoxia can skew a population's size distribution toward smaller individuals. These mechanisms produce sharp predictions about changes in seafood markets. Hypoxia is hypothesized to decrease the quantity of large shrimp relative to small shrimp and increase the price of large shrimp relative to small shrimp. We test these hypotheses using time series of size-based prices. Naive quantity-based models using treatment/control comparisons in hypoxic and nonhypoxic areas produce null results, but we find strong evidence of the hypothesized effects in the relative prices: Hypoxia increases the relative price of large shrimp compared with small shrimp. The effects of fuel prices provide supporting evidence. Empirical models of fishing effort and bioeconomic simulations explain why quantifying effects of hypoxia on fisheries using quantity data has been inconclusive. Specifically, spatial-dynamic feedbacks across the natural system (the fish stock) and human system (the mobile fishing fleet) confound "treated" and "control" areas. Consequently, analyses of price data, which rely on a market counterfactual, are able to reveal effects of the ecological disturbance that are obscured in quantity data. Our results are an important step toward quantifying the economic value of reduced upstream nutrient loading in the Mississippi Basin and are broadly applicable to other coupled human-natural systems.

  13. Seafood prices reveal impacts of a major ecological disturbance

    PubMed Central

    Smith, Martin D.; Oglend, Atle; Kirkpatrick, A. Justin; Asche, Frank; Bennear, Lori S.; Craig, J. Kevin; Nance, James M.

    2017-01-01

    Coastal hypoxia (dissolved oxygen ≤ 2 mg/L) is a growing problem worldwide that threatens marine ecosystem services, but little is known about economic effects on fisheries. Here, we provide evidence that hypoxia causes economic impacts on a major fishery. Ecological studies of hypoxia and marine fauna suggest multiple mechanisms through which hypoxia can skew a population’s size distribution toward smaller individuals. These mechanisms produce sharp predictions about changes in seafood markets. Hypoxia is hypothesized to decrease the quantity of large shrimp relative to small shrimp and increase the price of large shrimp relative to small shrimp. We test these hypotheses using time series of size-based prices. Naive quantity-based models using treatment/control comparisons in hypoxic and nonhypoxic areas produce null results, but we find strong evidence of the hypothesized effects in the relative prices: Hypoxia increases the relative price of large shrimp compared with small shrimp. The effects of fuel prices provide supporting evidence. Empirical models of fishing effort and bioeconomic simulations explain why quantifying effects of hypoxia on fisheries using quantity data has been inconclusive. Specifically, spatial-dynamic feedbacks across the natural system (the fish stock) and human system (the mobile fishing fleet) confound “treated” and “control” areas. Consequently, analyses of price data, which rely on a market counterfactual, are able to reveal effects of the ecological disturbance that are obscured in quantity data. Our results are an important step toward quantifying the economic value of reduced upstream nutrient loading in the Mississippi Basin and are broadly applicable to other coupled human-natural systems. PMID:28137850

  14. Synaptic transmission at parasympathetic neurons of the major pelvic ganglion from normal and diabetic male mice

    PubMed Central

    Vizzard, Margaret A.; Parsons, Rodney L.

    2013-01-01

    Bladder and erectile dysfunction are common urologic complications of diabetes and are associated with reduced parasympathetic autonomic control. To determine whether disruption of ganglionic neurotransmission contributes to the loss of function, we investigated synaptic transmission at parasympathetic, major pelvic ganglion (MPG) neurons in control and chronically (20 wk) diabetic mice. In contrast to what has been reported for sympathetic neurons, diabetes did not cause an interruption of synaptic transmission at parasympathetic MPG neurons from streptozotocin-treated C57BL/6J (STZ) or db/db mice. Cholinergically mediated excitatory postsynaptic potentials (EPSPs) were suprathreshold during 5-s trains of 5-, 10-, and 20-Hz stimuli. Asynchronous neurotransmitter release, observed as miniature EPSPs (mEPSPs) during and after stimulation, permitted quantitative assessment of postganglionic, cholinergic receptor sensitivity. mEPSP amplitude following tetanic stimulation (recorded at −60 mV) was reduced in STZ (4.95 ± 0.4 vs. 3.71 ± 0.3 mV, P = 0.03), but not db/db mice. The number of posttetanic mEPSPs was significantly greater in db/db mice at all frequencies tested. Assessment of basic electrophysiological properties revealed that parasympathetic MPG neurons from db/db mice had less negative membrane potentials, lower input resistances, and shorter afterhyperpolarizations relative to their control. MPG neurons from STZ had longer afterhyperpolarizations but were otherwise similar to controls. Membrane excitability, measured by the membrane responsiveness to long-duration (1 s), suprathreshold depolarizing pulses, was unchanged in either model. The present study indicates that, while parasympathetic neurotransmission at the MPG is intact in chronically diabetic mice, obese, type 2 diabetic animals exhibit an altered presynaptic regulation of neurotransmitter release. PMID:23197460

  15. Data on genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice.

    PubMed

    Grainger, Andrew T; Jones, Michael B; Li, Jing; Chen, Mei-Hua; Manichaikul, Ani; Shi, Weibin

    2016-12-01

    The data presented here are related to the research article, entitled Genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice, published in Atherosclerosis 2016;254:124 (A.T. Grainger, M.B. Jones, J. Li, M.H. Chen, A. Manichaikul, W. Shi, 2016) [1]. The supporting materials include original genotypic and phenotypic data obtained from 206 female F2 mice derived from an intercross between BALB and SMJ inbred mice. The F2 mice were fed 12 weeks of Western diet, starting at 6 weeks of age. Atherosclerotic lesion size in the aortic root of each mouse is the sum of the top 8 lesion areas. The data is provided in the format required for determining QTLs using two independent programs, J/QTL and PLINK.

  16. Prevention of intestinal obstruction reveals progressive neurodegeneration in mutant TDP-43 (A315T) mice

    PubMed Central

    2014-01-01

    Background Intraneuronal inclusions of TAR DNA-binding protein 43 (TDP-43) have been found in the majority of Amyotrophic Lateral Sclerosis (ALS) patients. Mutations in the gene encoding TDP-43 cause familial ALS. Transgenic mice expressing mutant TDP-43 with one such mutation (TDP-43 (A315T)) under control of the murine prion promoter develop motor symptoms, but their use is currently hampered by sudden death. We aimed to understand and overcome the cause of sudden death in TDP-43 (A315T) mice. Since intestinal obstruction was suspected to be the cause, intestinal motility of TDP-43 (A315T) mice was studied in an ex-vivo pellet propulsion assay. The effect on the enteric and motor phenotype was assessed, both in animals on normal chow or on a jellified fiber deprived diet, aimed at preventing intestinal obstruction. Results The frequency of the propulsive motor complexes was significantly reduced in the colon of TDP-43 (A315T) compared to non transgenic (NTG) mice. Immunohistochemistry revealed significant enlargement in size and reduction in number of the nitric oxide synthase (NOS) neurons in the myenteric plexus of TDP-43 (A315T) mice. Prevention of intestinal obstruction by jellified food abolished sudden death, allowing the motor phenotype to develop and slowly progress with a more pronounced degeneration of upper and lower motor axons. A downregulation of endogenous TDP-43 mRNA and protein levels was observed prior to neurodegeneration. Conclusion TDP-43 (A315T) mice suffer from intestinal dysmotility due to degeneration of NOS neurons in the myenteric plexus. Feeding the mice jellified food prevents sudden death and allows the motor phenotype to progress. PMID:24938805

  17. A fifth major genetic group among honeybees revealed in Syria

    PubMed Central

    2013-01-01

    Background Apiculture has been practiced in North Africa and the Middle-East from antiquity. Several thousand years of selective breeding have left a mosaic of Apis mellifera subspecies in the Middle-East, many uniquely adapted and survived to local environmental conditions. In this study we explore the genetic diversity of A. mellifera from Syria (n = 1258), Lebanon (n = 169) and Iraq (n = 35) based on 14 short tandem repeat (STR) loci in the context of reference populations from throughout the Old World (n = 732). Results Our data suggest that the Syrian honeybee Apis mellifera syriaca occurs in both Syrian and Lebanese territories, with no significant genetic variability between respective populations from Syria and Lebanon. All studied populations clustered within a new fifth independent nuclear cluster, congruent with an mtDNA Z haplotype identified in a previous study. Syrian honeybee populations are not associated with Oriental lineage O, except for sporadic introgression into some populations close to the Turkish and Iraqi borders. Southern Syrian and Lebanese populations demonstrated high levels of genetic diversity compared to the northern populations. Conclusion This study revealed the effects of foreign queen importations on Syrian bee populations, especially for the region of Tartus, where extensive introgression of A. m. anatolica and/or A. m. caucasica alleles were identified. The policy of creating genetic conservation centers for the Syrian subspecies should take into consideration the influence of the oriental lineage O from the northern Syrian border and the large population of genetically divergent indigenous honeybees located in southern Syria. PMID:24314104

  18. Major urinary metabolites of 6-keto-prostaglandin F2α in mice[S

    PubMed Central

    Kuklev, Dmitry V.; Hankin, Joseph A.; Uhlson, Charis L.; Hong, Yu H.; Murphy, Robert C.; Smith, William L.

    2013-01-01

    Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF2α urinary metabolites included dinor-6-keto-PGF2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases. PMID:23644380

  19. Transcriptional profiling reveals progeroid Ercc1-/Δ mice as a model system for glomerular aging

    PubMed Central

    2013-01-01

    Background Aging-related kidney diseases are a major health concern. Currently, models to study renal aging are lacking. Due to a reduced life-span progeroid models hold the promise to facilitate aging studies and allow examination of tissue-specific changes. Defects in genome maintenance in the Ercc1-/Δ progeroid mouse model result in premature aging and typical age-related pathologies. Here, we compared the glomerular transcriptome of young and aged Ercc1-deficient mice to young and aged WT mice in order to establish a novel model for research of aging-related kidney disease. Results In a principal component analysis, age and genotype emerged as first and second principal components. Hierarchical clustering of all 521 genes differentially regulated between young and old WT and young and old Ercc1-/Δ mice showed cluster formation between young WT and Ercc1-/Δ as well as old WT and Ercc1-/Δ samples. An unexpectedly high number of 77 genes were differentially regulated in both WT and Ercc1-/Δ mice (p < 0.0001). GO term enrichment analysis revealed these genes to be involved in immune and inflammatory response, cell death, and chemotaxis. In a network analysis, these genes were part of insulin signaling, chemokine and cytokine signaling and extracellular matrix pathways. Conclusion Beyond insulin signaling, we find chemokine and cytokine signaling as well as modifiers of extracellular matrix composition to be subject to major changes in the aging glomerulus. At the level of the transcriptome, the pattern of gene activities is similar in the progeroid Ercc1-/Δ mouse model constituting a valuable tool for future studies of aging-associated glomerular pathologies. PMID:23947592

  20. Characterization of Chronic Cutaneous Lesions from TNF-Receptor-1-Deficient Mice Infected by Leishmania major

    PubMed Central

    Oliveira, Carolina Ferreira; Manzoni-de-Almeida, Daniel; Mello, Paula Seixas; Natale, Caio Cotta; Santiago, Helton da Costa; Miranda, Luíza da Silva; Ferraz, Fernanda Oliveira; dos Santos, Liliane Martins; Teixeira, Mauro Martins; Arantes, Rosa Maria Esteves; Vieira, Leda Quercia

    2012-01-01

    Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis. PMID:22203861

  1. Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3.

    PubMed

    Wang, Xiaoming; McCoy, Portia A; Rodriguiz, Ramona M; Pan, Yanzhen; Je, H Shawn; Roberts, Adam C; Kim, Caroline J; Berrios, Janet; Colvin, Jennifer S; Bousquet-Moore, Danielle; Lorenzo, Isabel; Wu, Gangyi; Weinberg, Richard J; Ehlers, Michael D; Philpot, Benjamin D; Beaudet, Arthur L; Wetsel, William C; Jiang, Yong-Hui

    2011-08-01

    SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

  2. Immunomodulatory properties of borage (Echium amoenum) on BALB/c mice infected with Leishmania major.

    PubMed

    Hosseini, Nahid; Abolhassani, Mohsen

    2011-06-01

    Leishmaniasis is caused by parasitic protozoa transmitted by the bite of a female sand fly and is currently endemic in 88 countries. BALB/c mice are highly susceptible to the infection with the parasite Leishmania major, and this susceptibility has been attributed, in part, to the expansion of Th2 cells, production of their cytokines, and downregulation of Th1 cytokine, interferon gamma (IFN-γ). In this report, we used both aqueous and alcoholic extracts of Iranian borage (Echium amoenum Fisch & C.A. Mey) for treatment of L. major infection in BALB/c mice. We found that both extracts had immunomodulatory properties and increased the level of IFN-γ and lowered the parasite burden in the proximal lymph nodes and prevented the necrosis of the footpad as compared with the untreated infected mice. These results may provide a basis for further studies directed toward the use of the Iranian borage against L. major infection.

  3. An in vitro model for infection with Leishmania major that mimics the immune response in mice.

    PubMed Central

    Soares, M B; David, J R; Titus, R G

    1997-01-01

    By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development). PMID:9199457

  4. Establishment of Stable, Cell-Mediated Immunity that Makes "Susceptible" Mice Resistant to Leishmania major

    NASA Astrophysics Data System (ADS)

    Bretscher, Peter A.; Wei, Guojian; Menon, Juthika N.; Bielefeldt-Ohmann, Helle

    1992-07-01

    Cell-mediated, but not antibody-mediated, immune responses protect humans against certain pathogens that produce chronic diseases such as leishmaniasis. Effective vaccination against such pathogens must therefore produce an immunological "imprint" so that stable, cell-mediated immunity is induced in all individuals after natural infection. BALB/c mice "innately susceptible" to Leishmania major produce antibodies after substantial infection. In the present study, "susceptible" mice injected with a small number of parasites mounted a cell-mediated response and acquired resistance to a larger, normally pathogenic, challenge. This vaccination strategy may be applicable in diseases in which protection is dependent on cell-mediated immunity.

  5. Magnetic resonance imaging of urea transporter knockout mice reveals two distinct types of renal pelvic abnormality

    PubMed Central

    Jacob, Vinitha; Harbaugh, Calista; Dietz, John R.; Fenton, Robert A.; Kim, Soo Mi; Castrop, Hayo; Schnermann, Jurgen; Knepper, Mark A.; Chou, Chung-Lin; Anderson, Stasia A.

    2008-01-01

    Many transgenic and knockout mouse models with increased urine flow have been noted to have structural abnormalities of the renal pelvis and renal inner medulla. Here, we describe an approach for in vivo study of such abnormalities in mice using high resolution contrast enhanced T1-weighted magnetic resonance imaging (MRI). The studies were carried out in mice in which the UT-A isoform 1 and 3 urea transporters had been deleted (UT-A1/3-/- mice). The experiments revealed three distinct variations in the appearance of the renal pelvis in these mice: 1) normal kidneys with no accumulation of contrast agent in the renal pelvis; 2) frank right-sided unilateral hydronephrosis with marked atrophy of the renal medulla, seen relatively infrequently; and 3) a renal pelvic reflux pattern characterized by the presence of contrast agent in the renal pelvis surrounding the renal inner medulla, with no substantial atrophy of the renal medulla, seen in most UT-A1/3-/- mice with advancing age. The reflux pattern was also found in aquaporin-1 knockout mice. UT-A1/3-/- mice also manifested increased mean arterial pressure. Feeding the UT-A1/3-/- mice a low protein diet did not prevent the demonstrated abnormalities of the renal pelvis. These studies demonstrate the feasibility of real time imaging of renal pelvic structure in genetically manipulated mice, providing a tool for non-destructive, temporal studies of kidney structure. PMID:18854850

  6. Genetic variation and phylogeography of central Asian and other house mice, including a major new mitochondrial lineage in Yemen.

    PubMed Central

    Prager, E M; Orrego, C; Sage, R D

    1998-01-01

    The mitochondrial DNA (mtDNA) control region and flanking tRNAs were sequenced from 76 mice collected at 60 localities extending from Egypt through Turkey, Yemen, Iran, Afghanistan, Pakistan, and Nepal to eastern Asia. Segments of the Y chromosome and of a processed p53 pseudogene (Psip53) were amplified from many of these mice and from others collected elsewhere in Eurasia and North Africa. The 251 mtDNA types, including 54 new ones reported here, now identified from commensal house mice (Mus musculus group) by sequencing this segment can be organized into four major lineages-domesticus, musculus, castaneus, and a new lineage found in Yemen. Evolutionary tree analysis suggested the domesticus mtDNAs as the sister group to the other three commensal mtDNA lineages and the Yemeni mtDNAs as the next oldest lineage. Using this tree and the phylogeographic approach, we derived a new model for the origin and radiation of commensal house mice whose main features are an origin in west-central Asia (within the present-day range of M. domesticus) and the sequential spreading of mice first to the southern Arabian Peninsula, thence eastward and northward into south-central Asia, and later from south-central Asia to north-central Asia (and thence into most of northern Eurasia) and to southeastern Asia. Y chromosomes with and without an 18-bp deletion in the Zfy-2 gene were detected among mice from Iran and Afghanistan, while only undeleted Ys were found in Turkey, Yemen, Pakistan, and Nepal. Polymorphism for the presence of a Psip53 was observed in Georgia, Iran, Turkmenistan, Afghanistan, and Pakistan. Sequencing of a 128-bp Psip53 segment from 79 commensal mice revealed 12 variable sites and implicated >/=14 alleles. The allele that appeared to be phylogenetically ancestral was widespread, and the greatest diversity was observed in Turkey, Afghanistan, Pakistan, and Nepal. Two mice provided evidence for a second Psip53 locus in some commensal populations. PMID:9755213

  7. Expression and localization of cysteine sulfinate decarboxylase in major salivary glands of male mice.

    PubMed

    Liu, Shengnan; Liu, Ying; Ma, Qiwang; Cui, Sheng; Liu, Jiali

    2015-04-01

    Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in mammalian cells. It plays a significant role in cell development, nutrition, and survival, such as in the regulation of ion transport and osmoregulation. Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine. Recently, the synthesis of taurine has been observed in the central nervous system, kidney, liver, and muscle. However, the synthesis of taurine in the salivary glands has still not been described in detail. We have detected CSD expression in the major salivary glands of adult male mice by real-time polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence. In addition, we determined the content of taurine by high-performance liquid chromatography (HPLC). The results show that taurine is present in high concentrations in the major salivary glands of male mice. CSD messenger RNA (mRNA) and protein are expressed in the major salivary glands of male mice. The relative levels of CSD mRNA increase from the submandibular gland (SMG) to the sublingual gland (SLG) and parotid gland (PG), but the levels of the CSD protein are the opposite. The immunofluorescence results indicate that CSD is mainly located in the excretory ducts (EDs) and interlobular duct (IL) of SMG and ED in SLG, respectively. These results suggest that the major salivary glands of male mice produce taurine through the CSD pathway, and the synthesis of taurine might be related to sodium reabsorption in the salivary glands. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2011-02-01

    Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4(+) lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4(-) lymphocyte up to one month post-challenge suggesting that CD4(-) lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.

  9. Effects of prolonged moderate body deuteration on proliferative activity in major cell renewal systems in mice

    SciTech Connect

    Hodel, A.; Gebbers, J.-O.; Cottier, H.; Laissue, J.A.

    1982-06-07

    To evaluate the effects of prolonged moderate body deuteration on incorporation of tritiated thymidine (/sup 3/HTdR) into the DNA of major cell renewal systems, young adult mice were given drinking fluid containing 30 % heavy water for 7, 14, 21, 42 and 70 days. Control mice drank tap water. Three hours prior to sacrifice, 925 kBq of HTdR were injected intravenously. Following extraction of the bulk of the soluble activity with an aqueous formalin solution, the residual /sup 3/H-activity of the organs was assayed by liquid scintillation counting and by autoradiography. The total thymic /sup 3/H-activity and the thymic weight, particularly of the cortex, were significantly reduced indeuterated mice early in the course of the experiment. The fraction of labeled thymocytes diminished to less than one half of the control values on day 70. The /sup 3/H-activity of the bone marrow in deuterated mice was reduced to about 3.4 of control values. In contrast, the total /sup 3/H-activity of the small intestine, as well as mean labeling index and mitotic index of small intestinal epithelia, were mot markedly altered in deuterated versus control mice. Drinking water containing 30 % of heavy water did thus not result in generalized, profound and progressive disturbance of HTdR incorporation in all the major cell renewal systems in the mouse. The thymus and, to a lesser extent, the bone marrow, were unquestionably affected, but the depression of HTdR incorporation did not increase markedly in the course of 10 weeks. In these terms, the toxicity of 30 % heavy wateer in the drinking fluid appears minor. This is of particular interest since exposure to similar concentrations is known to elicit immunodepressive and/or antineoplastic effects.

  10. Ontogeny of Hepatic Energy Metabolism Genes in Mice as Revealed by RNA-Sequencing

    PubMed Central

    Renaud, Helen J.; Cui, Yue Julia; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2014-01-01

    The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age). The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5–Day 5 (perinatal-enriched), Day 10–Day 20 (pre-weaning-enriched), and Day 25–Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty acids-like 3

  11. Ontogeny of hepatic energy metabolism genes in mice as revealed by RNA-sequencing.

    PubMed

    Renaud, Helen J; Cui, Yue Julia; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D

    2014-01-01

    The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age). The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5-Day 5 (perinatal-enriched), Day 10-Day 20 (pre-weaning-enriched), and Day 25-Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty acids-like 3. These

  12. A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice.

    PubMed

    Tu, Jianxin; Hou, Bailong; Wang, Bingbing; Lin, Xiaoyun; Gong, Wenci; Dong, Haiyan; Zhu, Shanli; Chen, Shao; Xue, Xiangyang; Zhao, Kong-Nan; Zhang, Lifang

    2014-05-01

    We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) multi-epitope of Chlamydia trachomatis. A short gene of multi-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a(+) containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His-MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multi-epitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies (IgG1 and IgG2a), but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi-epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Th1 response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi-epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.

  13. Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice.

    PubMed

    Guiñazú, Natalia; Pellegrini, Andrea; Giordanengo, Laura; Aoki, Maria P; Rivarola, Hector W; Cano, Roxana; Rodrigues, Mauricio M; Gea, Susana

    2004-11-01

    BALB/c mice immunized with cruzipain, a major Trypanosoma cruzi antigen, produce specific and autoreactive immune responses against heart myosin, associated with cardiac functional and structural abnormalities. Preferential activation of the Th2 phenotype and an increase in cell populations expressing CD19+, Mac-1+ and Gr-1+ markers were found in the spleens of these mice. The aim of the present study was to investigate whether cardiac autoimmunity could be induced by cruzipain immunization of C57BL/6 mice and to compare the immune response elicited with that of BALB/c mice. We demonstrate that immune C57BL/6 splenocytes, re-stimulated in vitro with cruzipain, produced high levels of IFNgamma and low levels of IL-4 compatible with a Th1 profile. In contrast to BALB/c mice, spleens from cruzipain immune C57BL/6 mice revealed no significant changes in the number of cells presenting CD19+, Mac-1+ and Gr-1+ markers. An increased secretion of TGFbeta and a greater number of CD4+ TGFbeta+ cells were found in immune C57BL/6 but not in BALB/c mice. These findings were associated with the lack of autoreactive response against heart myosin and a myosin- or cruzipain-derived peptide. Thus, the differential immune response elicited in C57BL/6 and BALB/c mice upon cruzipain immunization is implicated in the resistance or pathogenesis of experimental Chagas' disease.

  14. Generation of Venus reporter knock-in mice revealed MAGI-2 expression patterns in adult mice.

    PubMed

    Ihara, Kan-ichiro; Nishimura, Tomoki; Fukuda, Tomokazu; Ookura, Tetsuya; Nishimori, Katsuhiko

    2012-01-01

    The membrane-associated guanylate kinase inverted 2 (MAGI-2) protein, which is known to localize at the tight junction of epithelial cells, contains multiple copies of the PDZ and WW domains in its structure. Although the expression pattern of Magi2 mRNA in representative organs has been previously published, its detailed cellular distribution at the histological level remains unknown. Such detailed information would be useful to clarify the biological function of MAGI-2. Here, we report the generation of Venus reporter knock-in mice for Magi2 in which exon 6 of the gene was substituted by the Venus-encoding sequence. We detected the expression of the Venus reporter protein in kidney podocytes from these knock-in mice. We also detected Venus reporter protein expression in spermatids within the testes and within neurons in various regions of the brain. Detection of the reporter protein from these diverse locations indicated the endogenous expression of MAGI-2 in these tissues. Our data suggested a potential function of MAGI-2 in the glomerular filtration process and sperm cell maturation. These data indicate that the Venus reporter knock-in mouse for Magi2 is a useful model for the further study of Magi2 gene function.

  15. Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome.

    PubMed

    Fok, Wilson C; Chen, Yidong; Bokov, Alex; Zhang, Yiqiang; Salmon, Adam B; Diaz, Vivian; Javors, Martin; Wood, William H; Zhang, Yongqing; Becker, Kevin G; Pérez, Viviana I; Richardson, Arlan

    2014-01-01

    Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.

  16. Mice Fed Rapamycin Have an Increase in Lifespan Associated with Major Changes in the Liver Transcriptome

    PubMed Central

    Fok, Wilson C.; Chen, Yidong; Bokov, Alex; Zhang, Yiqiang; Salmon, Adam B.; Diaz, Vivian; Javors, Martin; Wood, William H.; Zhang, Yongqing; Becker, Kevin G.; Pérez, Viviana I.; Richardson, Arlan

    2014-01-01

    Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity. PMID:24409289

  17. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  18. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  19. Effects of sertraline on the pharmacokinetics of bupropion and its major metabolite, hydroxybupropion, in mice.

    PubMed

    Molnari, Jillissa C; Hassan, Hazem E; Myers, Alan L

    2012-03-01

    Sertraline potently inhibits cytochrome P450 2B6 (CYP2B6) in vitro. Bupropion is commonly co-prescribed with sertraline and is exclusively metabolized by CYP2B6 to its major active metabolite hydroxybupropion. Putatively the co-administration of bupropion and sertraline could lead to a significant pharmacokinetic drug-drug interaction. The aim of this study was to evaluate a possible drug interaction between these drugs in mice. To study this male CF-1 mice were administered sertraline 5 mg/kg once daily for 6 days, followed by a single dose of bupropion 50 mg/kg on the seventh study day. Plasma and brain samples were collected post-bupropion dose for measurement of bupropion and hydroxybupropion levels on HPLC. Pharmacokinetic parameters for bupropion and hydroxybupropion were calculated using noncompartmental analysis and the variance in AUC of each was computed using Bailer's analysis. We found that mice pretreated with sertraline exhibited a small elevation in bupropion metabolism. This was substantiated by Bailer's analysis which indicated that in the presence of sertraline, both plasma and brain bupropion exposure were significantly (p < 0.05) decreased, while plasma hydroxybupropion exposure was significantly (p < 0.05) increased. Also the plasma hydroxybupropion-to-bupropion ratio of AUC was increased by 27% in sertraline treated mice, indicative of increased CYP2B activity. This is the first study, to our knowledge, that reports a mild pharmacokinetic drug-drug interaction between bupropion and sertraline in mice. However, it is unknown whether these quantitative changes in enzyme activity and consequent drug exposure would equate to significant pharmacodynamic changes (e.g., perturbations in brain neurotransmitter levels) observed in the clinic.

  20. Weak Proinsulin Peptide–Major Histocompatibility Complexes Are Targeted in Autoimmune Diabetes in Mice

    PubMed Central

    Levisetti, Matteo G.; Lewis, Danna M.; Suri, Anish; Unanue, Emil R.

    2008-01-01

    OBJECTIVE—Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes. RESEARCH DESIGN AND METHODS—The binding of a proinsulin epitope, proinsulin-1(47–64) (PI-1[47–64]), to the MHC class II molecules I-Ag7 and I-Ak was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in I-Ag7+ and I-Ak+ mice. RESULTS—C-peptide epitopes bound very weakly to I-Ag7 molecules. However, C-peptide–reactive T-cells were induced after immunization in I-Ag7–bearing mice (NOD and B6.g7) but not in I-Ak–bearing mice (B10.BR and NOD.h4). T-cells reactive with the PI-1(47–64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated β-cells in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice. CONCLUSIONS—These data demonstrate an inverse relationship between self-peptide–MHC binding and T-cell autoreactivity for the PI-1(47–64) epitope in autoimmune diabetes. PMID:18398138

  1. Experimental therapeutic studies of Solanum aculeastrum Dunal. on Leishmania major infection in BALB/c mice

    PubMed Central

    Laban, Linet T; Anjili, Christopher O; Mutiso, Joshua M; Ingonga, Johnstone; Kiige, Samuel G; Ngedzo, Mgala M; Gicheru, Michael M

    2015-01-01

    Objective(s): Solanum acueastrum Dunal. has been shown to have some chemotherapeutic value. Leaf and berry water and methanol compounds of S. acueastrum were evaluated for possible antileishmanial activity In vivo on BALB/c mice and in vitro against Leishmania major promastigotes, amastigotes and vero cells. Materials and Methods: Dry S. aculeastrum berry and leaf material were extracted in methanol and water. L. major parasites were exposed to different concentrations of S. aculeastrum fruit and leaf compounds and the IC50 on the promastigotes, percentage of infection rate of macrophages by amastigotes and the toxicological effect on vero cells were determined. BALB/c mice were infected subcutaneously with 1×106 promastigotes and kept for four weeks to allow for disease establishment. Infected mice were treated with fruit and leaf methanolic and water compounds, amphotericin B (AmB), and sterile phosphate buffered saline (PBS). Results: Fruit methanol compound was most effective in inhibiting the growth of promastigotes with IC5078.62 μg/ml. Fruit water compound showed the best activity in inhibiting infection of macrophages by amastigotes. Fruit methanol compound was more toxic at Ld50=8.06 mg/ml to vero cells than amphotericin B. Analysis of variance computation indicated statistically significant difference in lesion sizes between experimental and control mice groups (P=0.0001). Splenic impression smears ANOVA indicated a highly significant difference in parasitic numbers between the experimental and the control groups (P=0.0001). Conclusion: The results demonstrate that compounds from S. aculeastrum have potential anti-leishmanial activities and the medicinal use of the plant poses considerable toxicity against dividing vero cells. PMID:25810878

  2. Metabolomics reveals trichloroacetate as a major contributor to trichloroethylene-induced metabolic alterations in mouse urine and serum.

    PubMed

    Fang, Zhong-Ze; Krausz, Kristopher W; Tanaka, Naoki; Li, Fei; Qu, Aijuan; Idle, Jeffrey R; Gonzalez, Frank J

    2013-11-01

    Trichloroethylene (TCE)-induced liver toxicity and carcinogenesis is believed to be mediated in part by activation of the peroxisome proliferator-activated receptor α (PPARα). However, the contribution of the two TCE metabolites, dichloroacetate (DCA) and trichloroacetate (TCA) to the toxicity of TCE, remains unclear. The aim of the present study was to determine the metabolite profiles in serum and urine upon exposure of mice to TCE, to aid in determining the metabolic response to TCE exposure and the contribution of DCA and TCA to TCE toxicity. C57BL/6 mice were administered TCE, TCA, or DCA, and urine and serum subjected to ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based global metabolomics analysis. The ions were identified through searching metabolomics databases and by comparison with authentic standards, and quantitated using multiple reactions monitoring. Quantitative polymerase chain reaction of mRNA, biochemical analysis, and liver histology were also performed. TCE exposure resulted in a decrease in urine of metabolites involved in fatty acid metabolism, resulting from altered expression of PPARα target genes. TCE treatment also induced altered phospholipid homeostasis in serum, as revealed by increased serum lysophosphatidylcholine 18:0 and 18:1, and phosphatidylcholine metabolites. TCA administration revealed similar metabolite profiles in urine and serum upon TCE exposure, which correlated with a more robust induction of PPARα target gene expression associated with TCA than DCA treatment. These data show the metabolic response to TCE exposure and demonstrate that TCA is the major contributor to TCE-induced metabolite alterations observed in urine and serum.

  3. Mcph1-Deficient Mice Reveal a Role for MCPH1 in Otitis Media

    PubMed Central

    Chen, Jing; Ingham, Neil; Clare, Simon; Raisen, Claire; Vancollie, Valerie E.; Ismail, Ozama; McIntyre, Rebecca E.; Tsang, Stephen H.; Mahajan, Vinit B.; Dougan, Gordon; Adams, David J.; White, Jacqueline K.; Steel, Karen P.

    2013-01-01

    Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1tm1a/tm1a) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1tm1a/tm1a mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1tm1a/tm1a mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1tm1a/tm1a mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media. PMID:23516444

  4. Distinctive cellular immunity in genetically susceptible BALB/c mice recovered from Leishmania major infection or after subcutaneous immunization with killed parasites

    SciTech Connect

    Liew, F.Y.; Dhaliwal, J.S.

    1987-06-15

    Genetically susceptible BALB/c mice are refractory to further infection after recovery from Leishmania major infection after a sublethal dose of gamma-irradiation. In contrast, mice immunized with killed promastigotes s.c. develop exacerbated lesions after infection. Both groups of mice produce only a low level of specific antibody and no detectable cytotoxic T cells, but do have a strong antigen-specific DTH, which is adoptively transferable with Lyt-1+2-, L3T4+ T cells. Kinetic and histological studies revealed that mice immunized s.c. developed Jones-Mote hypersensitivity, peaking at 15 hr. with little mononuclear cell infiltration at the site of antigen administration; whereas mice that had recovered from infection developed tuberculin-type of reactivity, peaking at 24 to 48 hr, with intense mononuclear cell infiltration. Splenic T cells from recovered mice, when injected into the footpads of normal recipients together with live promastigotes, were able to retard lesion development; whereas T cells from s.c. immunized mice, when similarly transferred, accelerated disease progression. Antigen-specific culture supernatant of spleen cells from recovered mice also activated normal resident peritoneal macrophages to kill intracellular L. major amastigotes and tumor cells. Culture supernatants of spleen cells from s.c. immunized or normal mice were devoid of such activities. Part of the macrophage-activating potential can be inhibited by antibody specific for IFN-gamma. These results therefore demonstrate that whereas the Jones-Mote reaction is correlated with disease exacerbation, the tuberculin-type of DTH may be protective. Furthermore, in vivo immunity is directly related to the capacity of T cells to produce macrophage-activating factor.

  5. Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

    SciTech Connect

    Swelm, Rachel P.L. van; Laarakkers, Coby M.M.; Pertijs, Jeanne C.L.M.; Verweij, Vivienne; Masereeuw, Rosalinde; Russel, Frans G.M.

    2013-06-01

    Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75 mg/kg bw DF by oral gavage and 24 h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p < 0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p < 0.001 and p < 0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p < 0.001) and protein expression of PCNA (p < 0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24 h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury. - Highlights: • The urinary proteome shows biological processes involved in adverse drug reactions. • Urine proteins of DF-treated mice relate to kidney injury rather than liver injury. • Liver regeneration, not liver injury, is apparent 24h after oral DF administration. • Pretreatment with LPS does not enhance DF-induced liver injury in mice.

  6. Paeonol, a Major Compound of Moutan Cortex, Attenuates Cisplatin-Induced Nephrotoxicity in Mice

    PubMed Central

    Lee, Hyojung; Lee, Gihyun; Kim, Hyunseong; Bae, Hyunsu

    2013-01-01

    Cisplatin is an effective chemotherapeutic agent that is used for the treatment of a variety of cancers; however, its nephrotoxicity limits the use of this drug. In the present study, we examined whether paeonol, a major compound of Moutan Cortex, has protective effects on cisplatin-induced acute renal failure in mice. To accomplish this, Balb/c mice (6 to 8 wk of age, weighing 20 to 25 g) were administered, Moutan Cortex (300 mg/kg) or paeonol (20 mg/kg) once a day. At day 4, mice received cisplatin (30, 20, or 10 mg/kg) intraperitoneally. The paeonol-treated group showed marked attenuation of serum creatine and blood urea nitrogen levels as well as reduced levels of proinflammatory cytokines and nitric oxide when compared to the control group. In addition, the paeonol-treated group showed prolonged survival and marked attenuation of renal tissue injury. Taken together, these results demonstrated that paeonol can prevent the renal toxic effects of cisplatin. PMID:24171038

  7. CYP2D plays a major role in berberine metabolism in liver of mice and humans.

    PubMed

    Guo, Ying; Li, Feng; Ma, Xiaochao; Cheng, Xingguo; Zhou, Honghao; Klaassen, Curtis D

    2011-11-01

    Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.

  8. The major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.

    PubMed

    Bose, Mousumi; Lambert, Joshua D; Ju, Jihyeung; Reuhl, Kenneth R; Shapses, Sue A; Yang, Chung S

    2008-09-01

    In this study, we investigated the effects of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), on high-fat-induced obesity, symptoms of the metabolic syndrome, and fatty liver in mice. In mice fed a high-fat diet (60% energy as fat), supplementation with dietary EGCG treatment (3.2 g/kg diet) for 16 wk reduced body weight (BW) gain, percent body fat, and visceral fat weight (P < 0.05) compared with mice without EGCG treatment. The BW decrease was associated with increased fecal lipids in the high-fat-fed groups (r(2) = 0.521; P < 0.05). EGCG treatment attenuated insulin resistance, plasma cholesterol, and monocyte chemoattractant protein concentrations in high-fat-fed mice (P < 0.05). EGCG treatment also decreased liver weight, liver triglycerides, and plasma alanine aminotransferase concentrations in high-fat-fed mice (P < 0.05). Histological analyses of liver samples revealed decreased lipid accumulation in hepatocytes in mice treated with EGCG compared with high-fat diet-fed mice without EGCG treatment. In another experiment, 3-mo-old high-fat-induced obese mice receiving short-term EGCG treatment (3.2 g/kg diet, 4 wk) had decreased mesenteric fat weight and blood glucose compared with high-fat-fed control mice (P < 0.05). Our results indicate that long-term EGCG treatment attenuated the development of obesity, symptoms associated with the metabolic syndrome, and fatty liver. Short-term EGCG treatment appeared to reverse preexisting high-fat-induced metabolic pathologies in obese mice. These effects may be mediated by decreased lipid absorption, decreased inflammation, and other mechanisms.

  9. Transient receptor potential ankyrin-1 has a major role in mediating visceral pain in mice.

    PubMed

    Cattaruzza, Fiore; Spreadbury, Ian; Miranda-Morales, Marcela; Grady, Eileen F; Vanner, Stephen; Bunnett, Nigel W

    2010-01-01

    The excitatory ion channel transient receptor potential ankyrin-1 (TRPA1) is prominently expressed by primary afferent neurons and is a mediator of inflammatory pain. Inflammatory agents can directly activate [e.g., hydroxynonenal (HNE), prostaglandin metabolites] or indirectly sensitize [e.g., agonists of protease-activated receptor (PAR(2))] TRPA1 to induce somatic pain and hyperalgesia. However, the contribution of TRPA1 to visceral pain is unknown. We investigated the role of TRPA1 in visceral hyperalgesia by measuring abdominal visceromotor responses (VMR) to colorectal distention (CRD) after intracolonic administration of TRPA1 agonists [mustard oil (MO), HNE], sensitizing agents [PAR(2) activating peptide (PAR(2)-AP)], and the inflammatory agent trinitrobenzene sulfonic acid (TNBS) in trpa1(+/+) and trpa1(-/-) mice. Sensory neurons innervating the colon, identified by retrograde tracing, coexpressed immunoreactive TRPA1, calcitonin gene-related peptide, and substance P, expressed TRPA1 mRNA and responded to MO with depolarizing currents. Intracolonic MO and HNE increased VMR to CRD and induced immunoreactive c-fos in spinal neurons in trpa1+/+ but not in trpa1(-/-) mice. Intracolonic PAR(2)-AP induced mechanical hyperalgesia in trpa1+/+ but not in trpa1(-/-) mice. TNBS-induced colitis increased in VMR to CRD and induced c-fos in spinal neurons in trpa1(+/+) but not in trpa1(-/-) mice. Thus TRPA1 is expressed by colonic primary afferent neurons. Direct activation of TRPA1 causes visceral hyperalgesia, and TRPA1 mediates PAR(2)-induced hyperalgesia. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in the colon. Our results suggest that TRPA1 has a major role in visceral nociception and may be a therapeutic target for colonic inflammatory pain.

  10. Toxicogenomic analysis reveals profibrogenic effects of trichloroethylene in autoimmune-mediated cholangitis in mice.

    PubMed

    Kopec, Anna K; Sullivan, Bradley P; Kassel, Karen M; Joshi, Nikita; Luyendyk, James P

    2014-10-01

    Epidemiological studies suggest that exposure to environmental chemicals increases the risk of developing autoimmune liver disease. However, the identity of specific chemical perpetrators and the mechanisms whereby environmental chemicals modify liver disease is unclear. Previous studies link exposure to trichloroethylene (TCE) with the development of autoimmune liver disease and exacerbation of autoimmunity in lupus-prone MRL mice. In this study, we utilized NOD.c3c4 mice, which spontaneously develop autoimmune cholangitis bearing resemblance to some features of primary biliary cirrhosis. Nine-week-old female NOD.c3c4 mice were given TCE (0.5 mg/ml) or its vehicle (1% Cremophor-EL) in drinking water for 4 weeks. TCE had little effect on clinical chemistry, biliary cyst formation, or hepatic CD3+ T-cell accumulation. Hepatic microarray profiling revealed a dramatic suppression of early growth response 1 (EGR1) mRNA in livers of TCE-treated mice, which was verified by qPCR and immunohistochemical staining. Consistent with a reported link between reduced EGR1 expression and liver fibrosis, TCE increased hepatic type I collagen (COL1A1) mRNA and protein levels in livers of NOD.c3c4 mice. In contrast, TCE did not increase COL1A1 expression in NOD.ShiLtJ mice, which do not develop autoimmune cholangitis. These results suggest that in the context of concurrent autoimmune liver disease with a genetic basis, modification of hepatic gene expression by TCE may increase profibrogenic signaling in the liver. Moreover, these studies suggest that NOD.c3c4 mice may be a novel model to study gene-environment interactions critical for the development of autoimmune liver disease.

  11. Toxicogenomic Analysis Reveals Profibrogenic Effects of Trichloroethylene in Autoimmune-Mediated Cholangitis in Mice

    PubMed Central

    Kassel, Karen M.; Joshi, Nikita; Luyendyk, James P.

    2014-01-01

    Epidemiological studies suggest that exposure to environmental chemicals increases the risk of developing autoimmune liver disease. However, the identity of specific chemical perpetrators and the mechanisms whereby environmental chemicals modify liver disease is unclear. Previous studies link exposure to trichloroethylene (TCE) with the development of autoimmune liver disease and exacerbation of autoimmunity in lupus-prone MRL mice. In this study, we utilized NOD.c3c4 mice, which spontaneously develop autoimmune cholangitis bearing resemblance to some features of primary biliary cirrhosis. Nine-week-old female NOD.c3c4 mice were given TCE (0.5 mg/ml) or its vehicle (1% Cremophor-EL) in drinking water for 4 weeks. TCE had little effect on clinical chemistry, biliary cyst formation, or hepatic CD3+ T-cell accumulation. Hepatic microarray profiling revealed a dramatic suppression of early growth response 1 (EGR1) mRNA in livers of TCE-treated mice, which was verified by qPCR and immunohistochemical staining. Consistent with a reported link between reduced EGR1 expression and liver fibrosis, TCE increased hepatic type I collagen (COL1A1) mRNA and protein levels in livers of NOD.c3c4 mice. In contrast, TCE did not increase COL1A1 expression in NOD.ShiLtJ mice, which do not develop autoimmune cholangitis. These results suggest that in the context of concurrent autoimmune liver disease with a genetic basis, modification of hepatic gene expression by TCE may increase profibrogenic signaling in the liver. Moreover, these studies suggest that NOD.c3c4 mice may be a novel model to study gene-environment interactions critical for the development of autoimmune liver disease. PMID:25055964

  12. Human apolipoprotein E4 targeted replacement in mice reveals increased susceptibility to sleep disruption and intermittent hypoxia

    PubMed Central

    Kaushal, Navita; Ramesh, Vijay

    2012-01-01

    Intermittent hypoxia (IH) and sleep fragmentation (SF) are major manifestations of sleep apnea, a frequent condition in aging humans. Sleep perturbations are frequent in Alzheimer's disease (AD) and may underlie the progression of disease. We hypothesized that acute short-term IH, SF, and their combination (IH+SF) may reveal unique susceptibility in sleep integrity in a murine model of AD. The effects of acute IH, SF, and IH+SF on sleep architecture, delta power, sleep latency, and core body temperature were assessed in adult male human ApoE4-targeted replacement mice (hApoE4) and wild-type (WT) controls. Slow wave sleep (SWS) was significantly reduced, and rapid eye movement (REM) sleep was almost abolished during acute exposure to IH alone and IH+SF for 6 h in hApoE4, with milder effects in WT controls. Decreased delta power during SWS did not show postexposure rebound in hApoE4 unlike WT controls. IH and IH+SF induced hypothermia, which was more prominent in hApoE4 than WT controls. Mice subjected to SF also showed sleep deficits but without hypothermia. hApoE4 mice, unlike WT controls, exhibited increased sleep propensity, especially following IH and IH+SF, suggesting limited ability for sleep recovery in hApoE4 mice. These findings substantiate the potential impact of IH and SF in modulating sleep architecture and sleep homeostasis including maintenance of body temperature. Furthermore, the increased susceptibility and limited recovery ability of hApoE4 mice to sleep apnea suggests that early recognition and treatment of the latter in AD patients may restrict the progression and clinical manifestations of this frequent neurodegenerative disorder. PMID:22573105

  13. Conditional Knockout in Mice Reveals the Critical Roles of Ppp2ca in Epidermis Development.

    PubMed

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-05-18

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2ca(flox/flox) transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2ca(flox/flox); Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2ca(flox/flox); Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2ca(flox/flox) mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases.

  14. Analysis of intestinal microbiota in hybrid house mice reveals evolutionary divergence in a vertebrate hologenome.

    PubMed

    Wang, Jun; Kalyan, Shirin; Steck, Natalie; Turner, Leslie M; Harr, Bettina; Künzel, Sven; Vallier, Marie; Häsler, Robert; Franke, Andre; Oberg, Hans-Heinrich; Ibrahim, Saleh M; Grassl, Guntram A; Kabelitz, Dieter; Baines, John F

    2015-03-04

    Recent evidence suggests that natural selection operating on hosts to maintain their microbiome contributes to the emergence of new species, that is, the 'hologenomic basis of speciation'. Here we analyse the gut microbiota of two house mice subspecies, Mus musculus musculus and M. m. domesticus, across their Central European hybrid zone, in addition to hybrids generated in the lab. Hybrid mice display widespread transgressive phenotypes (that is, exceed or fall short of parental values) in a variety of measures of bacterial community structure, which reveals the importance of stabilizing selection operating on the intestinal microbiome within species. Further genetic and immunological analyses reveal genetic incompatibilities, aberrant immune gene expression and increased intestinal pathology associated with altered community structure among hybrids. These results provide unique insight into the consequences of evolutionary divergence in a vertebrate 'hologenome', which may be an unrecognized contributing factor to reproductive isolation in this taxonomic group.

  15. Analysis of intestinal microbiota in hybrid house mice reveals evolutionary divergence in a vertebrate hologenome

    PubMed Central

    Wang, Jun; Kalyan, Shirin; Steck, Natalie; Turner, Leslie M.; Harr, Bettina; Künzel, Sven; Vallier, Marie; Häsler, Robert; Franke, Andre; Oberg, Hans-Heinrich; Ibrahim, Saleh M.; Grassl, Guntram A.; Kabelitz, Dieter; Baines, John F.

    2015-01-01

    Recent evidence suggests that natural selection operating on hosts to maintain their microbiome contributes to the emergence of new species, that is, the ‘hologenomic basis of speciation’. Here we analyse the gut microbiota of two house mice subspecies, Mus musculus musculus and M. m. domesticus, across their Central European hybrid zone, in addition to hybrids generated in the lab. Hybrid mice display widespread transgressive phenotypes (that is, exceed or fall short of parental values) in a variety of measures of bacterial community structure, which reveals the importance of stabilizing selection operating on the intestinal microbiome within species. Further genetic and immunological analyses reveal genetic incompatibilities, aberrant immune gene expression and increased intestinal pathology associated with altered community structure among hybrids. These results provide unique insight into the consequences of evolutionary divergence in a vertebrate ‘hologenome’, which may be an unrecognized contributing factor to reproductive isolation in this taxonomic group. PMID:25737238

  16. Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner

    PubMed Central

    Mériaux, Véronique; Khan, Erin M.; Borde, Chloé; Ciulean, Ioana S.; Fitting, Catherine; Manoury, Bénédicte; Cavaillon, Jean-Marc; Doyen, Noëlle

    2016-01-01

    A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection. PMID:27182703

  17. In situ microscopy analysis reveals local innate immune response developed around Brucella infected cells in resistant and susceptible mice.

    PubMed

    Copin, Richard; Vitry, Marie-Alice; Hanot Mambres, Delphine; Machelart, Arnaud; De Trez, Carl; Vanderwinden, Jean-Marie; Magez, Stefan; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

    2012-01-01

    Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.

  18. Trends in genome dynamics among major orders of insects revealed through variations in protein families.

    PubMed

    Rappoport, Nadav; Linial, Michal

    2015-08-07

    Insects belong to a class that accounts for the majority of animals on earth. With over one million identified species, insects display a huge diversity and occupy extreme environments. At present, there are dozens of fully sequenced insect genomes that cover a range of habitats, social behavior and morphologies. In view of such diverse collection of genomes, revealing evolutionary trends and charting functional relationships of proteins remain challenging. We analyzed the relatedness of 17 complete proteomes representative of proteomes from insects including louse, bee, beetle, ants, flies and mosquitoes, as well as an out-group from the crustaceans. The analyzed proteomes mostly represented the orders of Hymenoptera and Diptera. The 287,405 protein sequences from the 18 proteomes were automatically clustered into 20,933 families, including 799 singletons. A comprehensive analysis based on statistical considerations identified the families that were significantly expanded or reduced in any of the studied organisms. Among all the tested species, ants are characterized by an exceptionally high rate of family gain and loss. By assigning annotations to hundreds of species-specific families, the functional diversity among species and between the major clades (Diptera and Hymenoptera) is revealed. We found that many species-specific families are associated with receptor signaling, stress-related functions and proteases. The highest variability among insects associates with the function of transposition and nucleic acids processes (collectively coined TNAP). Specifically, the wasp and ants have an order of magnitude more TNAP families and proteins relative to species that belong to Diptera (mosquitoes and flies). An unsupervised clustering methodology combined with a comparative functional analysis unveiled proteomic signatures in the major clades of winged insects. We propose that the expansion of TNAP families in Hymenoptera potentially contributes to the accelerated

  19. Complexity of gastric acid secretion revealed by targeted gene disruption in mice.

    PubMed

    Chen, Duan; Zhao, Chun-Mei

    2010-01-01

    Physiology of gastric acid secretion is one of the earliest subjects in medical research and education. Gastric acid secretion has been sometimes inadequately expressed as pH value rather than amount of gastric H(+) secreted per unit time. Gastric acid secretion is regulated by endocrine, paracrine and neurocrine signals via at least three messenger pathways: gastrin-histamine, CCK-somatostatin, and neural network. These pathways have been largely validated and further characterized by phenotyping a series of knockout mouse models. The complexity of gastric acid secretion is illustrated by both expected and unexpected phenotypes of altered acid secretion. For examples, in comparison with wild-type mice, gastrin and CCK double knockout and SSTR(2) knockout mice displayed a shift in the regulation of ECL cells from somatostatin-SSTR(2) pathway to galanin-Gal1 receptor pathway; a shift in the regulation of parietal cells from gastrin-histamine pathway to vagal pathway; and a shift in the CCK(2) receptors on parietal cells from functional silence to activation. The biological function of glycine-extended gastrin in synergizing gastrin-17 has been revealed in gastrin knockout mice. The roles of gastric acid secretion in tumorigenesis and ulceration have not been fully understood. Transgenic hypergastrinemic INS-GAS mice developed a spontaneous gastric cancer, which was associated with an impaired acid secretion. Gastrin knockout mice were still able to produce acid in response to vagal stimulation, especially after H. pylori infection. Taken together, phenotyping of a series of genetically engineered mouse models reveals a high degree of complexity of gastric acid secretion in both physiological and pathophysiological conditions.

  20. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection. PMID:27698715

  1. A quantitative framework for whole-body coordination reveals specific deficits in freely walking ataxic mice

    PubMed Central

    Machado, Ana S; Darmohray, Dana M; Fayad, João; Marques, Hugo G; Carey, Megan R

    2015-01-01

    The coordination of movement across the body is a fundamental, yet poorly understood aspect of motor control. Mutant mice with cerebellar circuit defects exhibit characteristic impairments in locomotor coordination; however, the fundamental features of this gait ataxia have not been effectively isolated. Here we describe a novel system (LocoMouse) for analyzing limb, head, and tail kinematics of freely walking mice. Analysis of visibly ataxic Purkinje cell degeneration (pcd) mice reveals that while differences in the forward motion of individual paws are fully accounted for by changes in walking speed and body size, more complex 3D trajectories and, especially, inter-limb and whole-body coordination are specifically impaired. Moreover, the coordination deficits in pcd are consistent with a failure to predict and compensate for the consequences of movement across the body. These results isolate specific impairments in whole-body coordination in mice and provide a quantitative framework for understanding cerebellar contributions to coordinated locomotion. DOI: http://dx.doi.org/10.7554/eLife.07892.001 PMID:26433022

  2. Congenic mice reveal genetic epistasis and overlapping disease loci for autoimmune diabetes and listeriosis.

    PubMed

    Wang, Nancy; Elso, Colleen M; Mackin, Leanne; Mannering, Stuart I; Strugnell, Richard A; Wijburg, Odilia L; Brodnicki, Thomas C

    2014-08-01

    The nonobese diabetic (NOD) mouse strain serves as a genomic standard for assessing how allelic variation for insulin-dependent diabetes (Idd) loci affects the development of autoimmune diabetes. We previously demonstrated that C57BL/6 (B6) mice harbor a more diabetogenic allele than NOD mice for the Idd14 locus when introduced onto the NOD genetic background. New congenic NOD mouse strains, harboring smaller B6-derived intervals on chromosome 13, now localize Idd14 to an ~18-Mb interval and reveal a new locus, Idd31. Notably, the B6 allele for Idd31 confers protection against diabetes, but only in the absence of the diabetogenic B6 allele for Idd14, indicating genetic epistasis between these two loci. Moreover, congenic mice that are more susceptible to diabetes are more resistant to Listeria monocytogenes infection. This result co-localizes Idd14 and Listr2, a resistance locus for listeriosis, to the same genomic interval and indicates that congenic NOD mice may also be useful for localizing resistance loci for infectious disease.

  3. Long-term home cage activity scans reveal lowered exploratory behaviour in symptomatic female Rett mice.

    PubMed

    Robinson, Lianne; Plano, Andrea; Cobb, Stuart; Riedel, Gernot

    2013-08-01

    Numerous experimental models have been developed to reiterate endophenotypes of Rett syndrome, a neurodevelopmental disorder with a multitude of motor, cognitive and vegetative symptoms. Here, female Mecp2(Stop) mice [1] were characterised at mild symptomatic conditions in tests for anxiety (open field, elevated plus maze) and home cage observation systems for food intake, locomotor activity and circadian rhythms. Aged 8-9 months, Mecp2(Stop) mice presented with heightened body weight, lower overall activity in the open field, but no anxiety phenotype. Although home cage activity scans conducted in two different observation systems, PhenoMaster and PhenoTyper, confirmed normal circadian activity, they revealed severely compromised habituation to a novel environment in all parameters registered including those derived from a non-linear decay model such as initial exploration maximum, decay half-life of activity and span, as well as plateau. Furthermore, overall activity was significantly reduced in nocturnal periods due to reductions in both fast ambulatory movements, but also a slow lingering. In contrast, light-period activity profiles during which the amount of sleep was highest remained normal in Mecp2(Stop) mice. These data confirm the slow and progressive development of Rett-like symptoms in female Mecp2(Stop) mice resulting in a prominent reduction of overall locomotor activity, while circadian rhythms are maintained. Alterations in the time-course of habituation may indicate deficiencies in cognitive processing.

  4. Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication.

    PubMed

    Achilli, Alessandro; Olivieri, Anna; Soares, Pedro; Lancioni, Hovirag; Hooshiar Kashani, Baharak; Perego, Ugo A; Nergadze, Solomon G; Carossa, Valeria; Santagostino, Marco; Capomaccio, Stefano; Felicetti, Michela; Al-Achkar, Walid; Penedo, M Cecilia T; Verini-Supplizi, Andrea; Houshmand, Massoud; Woodward, Scott R; Semino, Ornella; Silvestrelli, Maurizio; Giulotto, Elena; Pereira, Luísa; Bandelt, Hans-Jürgen; Torroni, Antonio

    2012-02-14

    Archaeological and genetic evidence concerning the time and mode of wild horse (Equus ferus) domestication is still debated. High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however, tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data reveal 18 major haplogroups (A-R) with radiation times that are mostly confined to the Neolithic and later periods and place the root of the phylogeny corresponding to the Ancestral Mare Mitogenome at ~130-160 thousand years ago. All haplogroups were detected in modern horses from Asia, but F was only found in E. przewalskii--the only remaining wild horse. Therefore, a wide range of matrilineal lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify well-preserved ancient remains, (ii) (re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the possible role of mtDNA backgrounds in racehorse performance.

  5. Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication

    PubMed Central

    Achilli, Alessandro; Olivieri, Anna; Soares, Pedro; Lancioni, Hovirag; Kashani, Baharak Hooshiar; Perego, Ugo A.; Nergadze, Solomon G.; Carossa, Valeria; Santagostino, Marco; Capomaccio, Stefano; Felicetti, Michela; Al-Achkar, Walid; Penedo, M. Cecilia T.; Verini-Supplizi, Andrea; Houshmand, Massoud; Woodward, Scott R.; Semino, Ornella; Silvestrelli, Maurizio; Giulotto, Elena; Pereira, Luísa; Bandelt, Hans-Jürgen; Torroni, Antonio

    2012-01-01

    Archaeological and genetic evidence concerning the time and mode of wild horse (Equus ferus) domestication is still debated. High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however, tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data reveal 18 major haplogroups (A–R) with radiation times that are mostly confined to the Neolithic and later periods and place the root of the phylogeny corresponding to the Ancestral Mare Mitogenome at ∼130–160 thousand years ago. All haplogroups were detected in modern horses from Asia, but F was only found in E. przewalskii—the only remaining wild horse. Therefore, a wide range of matrilineal lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify well-preserved ancient remains, (ii) (re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the possible role of mtDNA backgrounds in racehorse performance. PMID:22308342

  6. The role of "indirect" recognition in initiating rejection of skin grafts from major histocompatibility complex class II-deficient mice.

    PubMed Central

    Auchincloss, H; Lee, R; Shea, S; Markowitz, J S; Grusby, M J; Glimcher, L H

    1993-01-01

    In vitro studies have revealed several pathways by which T cells can respond to alloantigens, including CD4+ direct responses to allogeneic class II antigens, CD8+ direct responses to allogeneic class I antigens, and CD4+ "indirect" responses to peptides of alloantigens presented in association with responder class II molecules. In vivo studies of skin graft rejection, however, have so far provided clear evidence for the contribution of only the two direct pathways and not for indirect recognition. We have used major histocompatibility complex class II-deficient mice as donors to test the role of indirect recognition in rejection of skin grafts. Class II-deficient skin was always rejected without delay by normal recipients. Removal of recipient CD8+ cells (to leave the animals dependent on CD4+ function) or depletion of recipient CD4+ cells revealed that CD4+ cells were usually involved and sometimes absolutely required in this rapid rejection. Since the donor grafts lacked class II antigens, the CD4+ cells must have recognized donor antigens presented in association with recipient class II molecules. These results therefore indicate that indirect recognition can initiate rapid skin graft rejection. PMID:8475083

  7. Resting State fMRI in Mice Reveals Anesthesia Specific Signatures of Brain Functional Networks and Their Interactions

    PubMed Central

    Bukhari, Qasim; Schroeter, Aileen; Cole, David M.; Rudin, Markus

    2017-01-01

    fMRI studies in mice typically require the use of anesthetics. Yet, it is known that anesthesia alters responses to stimuli or functional networks at rest. In this work, we have used Dual Regression analysis Network Modeling to investigate the effects of two commonly used anesthetics, isoflurane and medetomidine, on rs-fMRI derived functional networks, and in particular to what extent anesthesia affected the interaction within and between these networks. Experimental data have been used from a previous study (Grandjean et al., 2014). We applied multivariate ICA analysis and Dual Regression to infer the differences in functional connectivity between isoflurane- and medetomidine-anesthetized mice. Further network analysis was performed to investigate within- and between-network connectivity differences between these anesthetic regimens. The results revealed five major networks in the mouse brain: lateral cortical, associative cortical, default mode, subcortical, and thalamic network. The anesthesia regime had a profound effect both on within- and between-network interactions. Under isoflurane anesthesia predominantly intra- and inter-cortical interactions have been observed, with only minor interactions involving subcortical structures and in particular attenuated cortico-thalamic connectivity. In contrast, medetomidine-anesthetized mice displayed subcortical functional connectivity including interactions between cortical and thalamic ICA components. Combining the two anesthetics at low dose resulted in network interaction that constituted the superposition of the interaction observed for each anesthetic alone. The study demonstrated that network modeling is a promising tool for analyzing the brain functional architecture in mice and comparing alterations therein caused by different physiological or pathological states. Understanding the differential effects of anesthetics on brain networks and their interaction is essential when interpreting fMRI data recorded under

  8. Resting State fMRI in Mice Reveals Anesthesia Specific Signatures of Brain Functional Networks and Their Interactions.

    PubMed

    Bukhari, Qasim; Schroeter, Aileen; Cole, David M; Rudin, Markus

    2017-01-01

    fMRI studies in mice typically require the use of anesthetics. Yet, it is known that anesthesia alters responses to stimuli or functional networks at rest. In this work, we have used Dual Regression analysis Network Modeling to investigate the effects of two commonly used anesthetics, isoflurane and medetomidine, on rs-fMRI derived functional networks, and in particular to what extent anesthesia affected the interaction within and between these networks. Experimental data have been used from a previous study (Grandjean et al., 2014). We applied multivariate ICA analysis and Dual Regression to infer the differences in functional connectivity between isoflurane- and medetomidine-anesthetized mice. Further network analysis was performed to investigate within- and between-network connectivity differences between these anesthetic regimens. The results revealed five major networks in the mouse brain: lateral cortical, associative cortical, default mode, subcortical, and thalamic network. The anesthesia regime had a profound effect both on within- and between-network interactions. Under isoflurane anesthesia predominantly intra- and inter-cortical interactions have been observed, with only minor interactions involving subcortical structures and in particular attenuated cortico-thalamic connectivity. In contrast, medetomidine-anesthetized mice displayed subcortical functional connectivity including interactions between cortical and thalamic ICA components. Combining the two anesthetics at low dose resulted in network interaction that constituted the superposition of the interaction observed for each anesthetic alone. The study demonstrated that network modeling is a promising tool for analyzing the brain functional architecture in mice and comparing alterations therein caused by different physiological or pathological states. Understanding the differential effects of anesthetics on brain networks and their interaction is essential when interpreting fMRI data recorded under

  9. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2007-06-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.

  10. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

    PubMed Central

    Kiaei, Simin Sadat; Iravani, Davood

    2007-01-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response. PMID:17570972

  11. Effects of the Activation of Three Major Hepatic Akt Substrates on Glucose Metabolism in Male Mice.

    PubMed

    Sakai, Gota; Inoue, Ikuo; Suzuki, Tokuko; Sumita, Takashi; Inukai, Kouichi; Katayama, Shigehiro; Awata, Takuya; Yamada, Tetsuya; Asano, Tomoichiro; Katagiri, Hideki; Noda, Mitsuhiko; Shimada, Akira; Ono, Hiraku

    2017-08-01

    Insulin suppresses glucose output from the liver via Akt activation; however, which substrate of Akt plays the major role in transducing this effect is unclear. We tested the postnatal expression of Akt-unresponsive, constitutively active mutants of three major Akt substrates widely considered to regulate glucose metabolism [i.e., FoxO1, PGC1α, and glycogen synthase kinase-3β (GSK3β)] using adenoviral gene delivery to the mouse liver. We performed physiological hyperinsulinemic-euglycemic clamp studies using these mice under awake and nonrestrained conditions with blood sampling via an arterial catheter. Hepatic expression of constitutively active FoxO1 induced significant hepatic and systemic insulin resistance. However, neither the expression of constitutively active PGC1α nor that of GSK3β significantly changed insulin sensitivity. Simultaneous expression of all three mutants together induced no further insulin resistance compared with that of the FoxO1 mutant. The glycogen content in the liver was significantly reduced by constitutively active GSK3β expression. In cultured hepatocytes, constitutively active PGC1α induced markedly stronger transcriptional enhancement of gluconeogenic key enzymes than did constitutively active FoxO1. From these results, we conclude that FoxO1 has the most prominent role in transducing insulin's effect downstream from Akt to suppress hepatic glucose output, involving mechanisms independent of the transcriptional regulation of key gluconeogenic enzymes. Copyright © 2017 Endocrine Society.

  12. Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self- major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells

    PubMed Central

    1981-01-01

    To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus- specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC- restricted CTL. By applying the limiting-dilution technique, the CTL- precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC- restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC- restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products. PMID:6265587

  13. Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT(-/-) mice.

    PubMed

    Stockebrand, Malte; Nejad, Ali Sasani; Neu, Axel; Kharbanda, Kusum K; Sauter, Kathrin; Schillemeit, Stefan; Isbrandt, Dirk; Choe, Chi-Un

    2016-08-01

    Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

  14. Protective response to Leishmania major in BALB/c mice requires antigen processing in the absence of DM.

    PubMed

    Kamala, Tirumalai; Nanda, Navreet K

    2009-04-15

    Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158-173. Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immunodominant LACK 158-173 on infected APC. In its place, infected DM(-/-) hosts show elicitation of CD4 T cells specific for newer epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM(-/-) mice is dependent on IFN-gamma. DM is thus a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens.

  15. Piezo2 is the major transducer of mechanical forces for touch sensation in mice

    PubMed Central

    Ranade, Sanjeev S.; Woo, Seung-Hyun; Dubin, Adrienne E.; Moshourab, Rabih A.; Wetzel, Christiane; Petrus, Matt; Mathur, Jayanti; Bégay, Valérie; Coste, Bertrand; Mainquist, James; Wilson, A.J.; Francisco, Allain G.; Reddy, Kritika; Qiu, Zhaozhu; Wood, John N.; Lewin, Gary R.; Patapoutian, Ardem

    2015-01-01

    Summary The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals1. It is postulated that mechanically activated (MA) cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive2. Piezo2 is a rapidly adapting (RA) MA ion channel expressed in a subset of sensory neurons of the dorsal root ganglion (DRG) and in cutaneous mechanoreceptors known as Merkel cell-neurite complexes3,4. Merkel cells have been demonstrated to play a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by its innervating sensory neuron4-6. However, major aspects of touch sensation remain intact without Merkel cell activity4,7. Here, we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low threshold mechanoreceptors (LTMRs) that innervate both hairy and glabrous skin. Most RA MA currents in DRG neuronal cultures are absent in Piezo2CKO mice, and ex vivo skin nerve preparation studies show that mechanosensitivity of LTMRs strongly depends on Piezo2. This striking cellular phenotype correlates with an unprecedented behavioral phenotype: an almost complete deficit in light touch sensation in multiple behavioral assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays RA MA currents in vitro is responsible for the mechanosensitivity of most LTMR subtypes involved in innocuous touch sensation. Interestingly, we find that touch and pain sensation are separable, suggesting that yet-unknown MA ion channel(s) must account for noxious (painful) mechanosensation. PMID:25471886

  16. Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

    PubMed

    Ranade, Sanjeev S; Woo, Seung-Hyun; Dubin, Adrienne E; Moshourab, Rabih A; Wetzel, Christiane; Petrus, Matt; Mathur, Jayanti; Bégay, Valérie; Coste, Bertrand; Mainquist, James; Wilson, A J; Francisco, Allain G; Reddy, Kritika; Qiu, Zhaozhu; Wood, John N; Lewin, Gary R; Patapoutian, Ardem

    2014-12-04

    The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

  17. Biogenic non-crystalline U(IV) revealed as major component in uranium ore deposits

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Amrita; Campbell, Kate M.; Kelly, Shelly D.; Roebbert, Yvonne; Weyer, Stefan; Bernier-Latmani, Rizlan; Borch, Thomas

    2017-06-01

    Historically, it is believed that crystalline uraninite, produced via the abiotic reduction of hexavalent uranium (U(VI)) is the dominant reduced U species formed in low-temperature uranium roll-front ore deposits. Here we show that non-crystalline U(IV) generated through biologically mediated U(VI) reduction is the predominant U(IV) species in an undisturbed U roll-front ore deposit in Wyoming, USA. Characterization of U species revealed that the majority (~58-89%) of U is bound as U(IV) to C-containing organic functional groups or inorganic carbonate, while uraninite and U(VI) represent only minor components. The uranium deposit exhibited mostly 238U-enriched isotope signatures, consistent with largely biotic reduction of U(VI) to U(IV). This finding implies that biogenic processes are more important to uranium ore genesis than previously understood. The predominance of a relatively labile form of U(IV) also provides an opportunity for a more economical and environmentally benign mining process, as well as the design of more effective post-mining restoration strategies and human health-risk assessment.

  18. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  19. Biogenic non-crystalline U(IV) revealed as major component in uranium ore deposits

    DOE PAGES

    Bhattacharyya, Amrita; Campbell, Kate M.; Kelly, Shelly D.; ...

    2017-06-01

    Historically, it is believed that crystalline uraninite, produced via the abiotic reduction of hexavalent uranium (U(VI)) is the dominant reduced U species formed in low-temperature uranium roll-front ore deposits. Here we show that non-crystalline U(IV) generated through biologically mediated U(VI) reduction is the predominant U(IV) species in an undisturbed U roll-front ore deposit in Wyoming, USA. Characterization of U species revealed that the majority (~58-89%) of U is bound as U(IV) to C-containing organic functional groups or inorganic carbonate, while uraninite and U(VI) represent only minor components. The uranium deposit exhibited mostly 238U-enriched isotope signatures, consistent with largely biotic reductionmore » of U(VI) to U(IV). This finding implies that biogenic processes are more important to uranium ore genesis than previously understood. The predominance of a relatively labile form of U(IV) also provides an opportunity for a more economical and environmentally benign mining process, as well as the design of more effective post-mining restoration strategies and human health-risk assessment.« less

  20. mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe.

    PubMed Central

    Torroni, A; Bandelt, H J; D'Urbano, L; Lahermo, P; Moral, P; Sellitto, D; Rengo, C; Forster, P; Savontaus, M L; Bonné-Tamir, B; Scozzari, R

    1998-01-01

    mtDNA sequence variation was studied in 419 individuals from nine Eurasian populations, by high-resolution RFLP analysis, and it was followed by sequencing of the control region of a subset of these mtDNAs and a detailed survey of previously published data from numerous other European populations. This analysis revealed that a major Paleolithic population expansion from the "Atlantic zone" (southwestern Europe) occurred 10,000-15,000 years ago, after the Last Glacial Maximum. As an mtDNA marker for this expansion we identified haplogroup V, an autochthonous European haplogroup, which most likely originated in the northern Iberian peninsula or southwestern France at about the time of the Younger Dryas. Its sister haplogroup, H, which is distributed throughout the entire range of Caucasoid populations and which originated in the Near East approximately 25,000-30,000 years ago, also took part in this expansion, thus rendering it by far the most frequent (40%-60%) haplogroup in western Europe. Subsequent migrations after the Younger Dryas eventually carried those "Atlantic" mtDNAs into central and northern Europe. This scenario, already implied by archaeological records, is given overwhelming support from both the distribution of the autochthonous European Y chromosome type 15, as detected by the probes 49a/f, and the synthetic maps of nuclear data. PMID:9545392

  1. Biogenic non-crystalline U(IV) revealed as major component in uranium ore deposits

    PubMed Central

    Bhattacharyya, Amrita; Campbell, Kate M.; Kelly, Shelly D.; Roebbert, Yvonne; Weyer, Stefan; Bernier-Latmani, Rizlan; Borch, Thomas

    2017-01-01

    Historically, it is believed that crystalline uraninite, produced via the abiotic reduction of hexavalent uranium (U(VI)) is the dominant reduced U species formed in low-temperature uranium roll-front ore deposits. Here we show that non-crystalline U(IV) generated through biologically mediated U(VI) reduction is the predominant U(IV) species in an undisturbed U roll-front ore deposit in Wyoming, USA. Characterization of U species revealed that the majority (∼58-89%) of U is bound as U(IV) to C-containing organic functional groups or inorganic carbonate, while uraninite and U(VI) represent only minor components. The uranium deposit exhibited mostly 238U-enriched isotope signatures, consistent with largely biotic reduction of U(VI) to U(IV). This finding implies that biogenic processes are more important to uranium ore genesis than previously understood. The predominance of a relatively labile form of U(IV) also provides an opportunity for a more economical and environmentally benign mining process, as well as the design of more effective post-mining restoration strategies and human health-risk assessment. PMID:28569759

  2. Biogenic non-crystalline U(IV) revealed as major component in uranium ore deposits

    USGS Publications Warehouse

    Bhattacharyya, Amrita; Campbell, Kate M.; Kelly, Shelly; Roebbert, Yvonne; Weyer, Stefan; Bernier-Latmani, Rizlan; Borch, Thomas

    2017-01-01

    Historically, it is believed that crystalline uraninite, produced via the abiotic reduction of hexavalent uranium (U(VI)) is the dominant reduced U species formed in low-temperature uranium roll-front ore deposits. Here we show that non-crystalline U(IV) generated through biologically mediated U(VI) reduction is the predominant U(IV) species in an undisturbed U roll-front ore deposit in Wyoming, USA. Characterization of U species revealed that the majority (∼58-89%) of U is bound as U(IV)to C-containing organic functional groups or inorganic carbonate, while uraninite and U(VI) represent only minor components. The uranium deposit exhibited mostly 238U-enriched isotope signatures, consistent with largely biotic reduction of U(VI) to U(IV). This finding implies that biogenic processes are more important to uranium ore genesis than previously understood. The predominance of a relatively labile form of U(IV) also provides an opportunity for a more economical and environmentally benign mining process, as well as the design of more effective post-mining restoration strategies and human health-risk assessment.

  3. Genetic signature of histiocytic sarcoma revealed by a sleeping beauty transposon genetic screen in mice.

    PubMed

    Been, Raha A; Linden, Michael A; Hager, Courtney J; DeCoursin, Krista J; Abrahante, Juan E; Landman, Sean R; Steinbach, Michael; Sarver, Aaron L; Largaespada, David A; Starr, Timothy K

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients.

  4. The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice.

    PubMed Central

    Esther, C R; Marino, E M; Howard, T E; Machaud, A; Corvol, P; Capecchi, M R; Bernstein, K E

    1997-01-01

    Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney. PMID:9153279

  5. Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

    PubMed Central

    Been, Raha A.; Linden, Michael A.; Hager, Courtney J.; DeCoursin, Krista J.; Abrahante, Juan E.; Landman, Sean R.; Steinbach, Michael; Sarver, Aaron L.; Largaespada, David A.; Starr, Timothy K.

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients. PMID:24827933

  6. Managing major data of genetically modified mice: from scientific demands to legal obligations.

    PubMed

    Staudt, Michael; Trauth, Jürgen; Hindi, Iris El; Galuschka, Claudia; Sitek, Dagmar; Schenkel, Johannes

    2012-10-01

    The number of genetically modified mice is increasing rapidly. Several limitations when working with these animals are to be considered: small colonies, the continued danger of loss, often a limited breeding-success, the need to keep those mutants in stock, difficult and costly import-procedures, and also a major (scientific) value of those mutants often available only with major restrictions. To gather relevant information about all active and archived genetically modified mouse lines available in-house (>1.500) and to deal with a unique resource for several, quite different purposes, a data base was developed enabling optimum knowledge management and easy access. The data base covers also legal restraints and is being linked with the institutional publication repository. To identify the lines available detailed information is provided for each line, as the international designation, a short name, the characterization/description, and the genetic modification including the technique used therefore. The origin of the mutation (gene-ID# and donor organism), the origin of regulatory elements and their donors are listed as well as the genetic background, back-cross generation, phenotype, possible publications, keywords, and some in-house information. Also aspects of animal welfare, obligations to record genetically modified organisms, and technology transfer are displayed; the latter to make licenses possible (if legally permitted). Material transfer agreements, patents, or legal restrictions are listed. This data base helps to avoid double-imports, saves animals and costs since a redundant generation or import can be omitted. However, this is a contribution to the 3R principles developed by Russell and Burch.

  7. Major urinary metabolites of 6-keto-prostaglandin F2α in mice.

    PubMed

    Kuklev, Dmitry V; Hankin, Joseph A; Uhlson, Charis L; Hong, Yu H; Murphy, Robert C; Smith, William L

    2013-07-01

    Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF2α urinary metabolites included dinor-6-keto-PGF2α (~10%) and dinor-13,14-dihydro-6,15-diketo-PGF1α (~10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.

  8. Comprehensive Plasma Metabolomic Analyses of Atherosclerotic Progression Reveal Alterations in Glycerophospholipid and Sphingolipid Metabolism in Apolipoprotein E-deficient Mice

    PubMed Central

    Dang, Vi T.; Huang, Aric; Zhong, Lexy H.; Shi, Yuanyuan; Werstuck, Geoff H.

    2016-01-01

    Atherosclerosis is the major underlying cause of most cardiovascular diseases. Despite recent advances, the molecular mechanisms underlying the pathophysiology of atherogenesis are not clear. In this study, comprehensive plasma metabolomics were used to investigate early-stage atherosclerotic development and progression in chow-fed apolipoprotein E-deficient mice at 5, 10 and 15 weeks of age. Comprehensive plasma metabolomic profiles, based on 4365 detected metabolite features, differentiate atherosclerosis-prone from atherosclerosis-resistant models. Metabolites in the sphingomyelin pathway were significantly altered prior to detectable lesion formation and at all subsequent time-points. The cytidine diphosphate-diacylglycerol pathway was up-regulated during stage I of atherosclerosis, while metabolites in the phosphatidylethanolamine and glycosphingolipid pathways were augmented in mice with stage II lesions. These pathways, involving glycerophospholipid and sphingolipid metabolism, were also significantly affected during the course of atherosclerotic progression. Our findings suggest that distinct plasma metabolomic profiles can differentiate the different stages of atherosclerotic progression. This study reveals that alteration of specific, previously unreported pathways of glycerophospholipid and sphingolipid metabolism are associated with atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. PMID:27721472

  9. Reconstruction of major fibers using 7T multi-shell Hybrid Diffusion Imaging in mice

    NASA Astrophysics Data System (ADS)

    Daianu, Madelaine; Jacobs, Russell E.; Zlokovic, Berislav V.; Montagne, Axel; Thompson, Paul M.

    2015-12-01

    Diffusion weighted imaging (DWI) can reveal the orientation of the underlying fiber populations in the brain. High angular resolution diffusion imaging (HARDI) is increasingly used to better resolve the orientation and mixing of fibers. Here, we assessed the added value of multi-shell q-space sampling on the reconstruction of major fibers using mathematical frameworks from q-ball imaging (QBI) and generalized q-sampling imaging (GQI), as compared to diffusion tensor imaging (DTI). We scanned a healthy mouse brain using 7-Tesla 5-shell HARDI (b=1000, 3000, 4000, 8000, 12000 s/mm2), also known as hybrid diffusion imaging (HYDI). We found that QBI may provide greater reconstruction accuracy for major fibers, which improves with the addition of higher b-value shells, unlike GQI or DTI (as expected). Although QBI is a special case of GQI, the major fiber orientation in QBI was more closely related to the orientation in DTI, rather than GQI. HYDI can aid the clinical outcomes of research and especially - more advanced human and animal connectomics projects to map the brain's neural pathways and networks.

  10. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    PubMed Central

    2010-01-01

    Background Because of their functional significance, the Major Histocompatibility Complex (MHC) class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective, genotyping of MHC systems of

  11. Re-evaluation of batoid pectoral morphology reveals novel patterns of diversity among major lineages.

    PubMed

    Martinez, Christopher M; Rohlf, F James; Frisk, Michael G

    2016-04-01

    Batoids (Chondrichthyes: Batoidea) are a diverse group of cartilaginous fishes which comprise a monophyletic sister lineage to all neoselachians or modern sharks. All species in this group possess anteroposteriorly expanded-pectoral fins, giving them a unique disc-like body form. Reliance on pectoral fins for propulsion ranges from minimal (sawfish) to almost complete dependence (skates and rays). A recent study on the diversity of planform pectoral fin shape in batoids compared overall patterns of morphological variation within the group. However, inconsistent pectoral homology prevented the study from accurately representing relationships within and among major batoid taxa. With previous work in mind, we undertook an independent investigation of pectoral form in batoids and evaluated the implications of shape diversity on locomotion and lifestyle, particularly in the skates (Rajoidei) and rays (Myliobatoidei). We used geometric morphometrics with sliding semilandmarks to analyze pectoral fin outlines and also calculate fin aspect ratios (AR), a functional trait linked to locomotion. In agreement with previous work, our results indicated that much of the evolution of batoid pectoral shape has occurred along a morphological axis that is closely related to AR. For species where kinematic data were available, both shape and AR were associated with swimming mode. This work further revealed novel patterns of shape variation among batoids, including strong bimodality of shape in rays, an intermediate location of skate species in the morphospace between benthic/demersal and pelagic rays, and approximately parallel shape trajectories in the benthic/demersal rays and skates. Finally, manipulation of landmarks verified the need for a consistent and accurate definition of homology for the outcome and efficacy of analyses of pectoral form and function in batoids.

  12. Therapeutic Effect of Hedera helix Alcoholic Extract Against Cutaneous Leishmaniasis Caused by Leishmania major in Balb/c Mice

    PubMed Central

    Hooshyar, Hossein; Talari, Safarali; Feyzi, Fatemeh

    2014-01-01

    Background: Cutaneous leishmaniasis (CL) is common and endemic in many areas of Iran, caused by species of a protozoan parasite belonging to the genus Leishmania. There is not any effective vaccine against leishmaniasis; so, therapy is important for prevention and separation of disease. Herbal extract for treatment of CL is cost-effective, applicable topically to lesions, and can avoid the development of drug resistance. Objectives: The aim of this study was to evaluate the in vivo activity of an alcoholic extract of Hedera helix (a native Iranian plant) on the experimental ulcer of zoonotic CL in Balb/c mice. Materials and Methods: At least 5x l06 promastigotes of Leishmania major (MHOM/64/IR/ER75) were inoculated subcutaneously in the tail base of Balb/c mice. Fifty six infected mice were distributed in four groups, two groups (16 mice for 20% alcoholic extract of H. helix and 13 for 70% extract) were used as experimental groups, one (15 mice) as placebo control (Control A), and one (12 mice) as negative control. Treatment effects of two concentrations were determined by comparison of placebo and nontreated groups via measuring the size of skin lesions and the number of parasitologically positive and negative mice after the therapy period. Results: This study showed that the main lesion size did not decrease significantly, or the small lesions did not completely disappear after treatment by H. helix alcoholic extract. Amastigotes counts (mean ± SD) of the skin lesions decreased in control A and 20% concentration groups, but in negative control and 70% concentration groups the number of parasites did not reduce. Conclusions: The present study did not support the in vivo antileishmanial effect of H. helix extract. We recommend further studies using major components of H. helix, especially hederasaponin (saponin K10), to investigate the antileishmanial effect of this plant on L. major. PMID:25147703

  13. Role of major histocompatibility complex class II in resistance of mice to naturally acquired infection with Syphacia obvelata

    NASA Technical Reports Server (NTRS)

    Stewart, Patricia W.; Chapes, Stephen K.

    2003-01-01

    Genetics plays a substantial role in host resistance in many host-parasite interactions. We examined the prevalence of naturally acquired infection with Syphacia obvelata in a number of mouse strains housed in a non-barrier facility. These mice, which included cross-bred and congenic, inbred strains on various genetic backgrounds, differ in the loci for the immune function genes--major histocompatibility complex class II (MHCII), toll-like receptor 4 (Tlr4), and solute carrier family 11, member 1 (Slc11a1)--which allowed comparisons of the impact of these genes on resistance to pinworm infection. Male and female mice of various ages were sampled over an 18-month period; infection was determined by use of the cellophane tape test. Results indicated that mice that were MHCII+/+ had a significantly lower prevalence of infection than did mice that were MHCII-/-. Differences were not seen between male and female mice. Although MHCII+/+ mice had an age-associated decrease in infection prevalence, such decrease was not seen in MHCII-/- mice. In contrast, infection prevalence in mice with the normal Tlr4 gene (Tlr4(LPS-n/LPS-n)) gene did not differ significantly compared with that in mice that were homozygous for either the point mutation (Tlr4(LPS-d/LPS-d)) or deletion (Tlr4(LPS-del/LPS-del)) of that gene. Likewise, the presence (Sle11a1r/r) or absence (Slc11a1s/s) of functional alleles for Slc11a1 had no effect on the prevalence of infection with S. obvelata. In conclusion, presence of MHCII, but not Tlr4 or Slc11a1 significantly influences prevalence of naturally acquired infection with S. obvelata. These data justify further comprehensive analyses of the immune components that are involved in pinworm resistance.

  14. Role of major histocompatibility complex class II in resistance of mice to naturally acquired infection with Syphacia obvelata

    NASA Technical Reports Server (NTRS)

    Stewart, Patricia W.; Chapes, Stephen K.

    2003-01-01

    Genetics plays a substantial role in host resistance in many host-parasite interactions. We examined the prevalence of naturally acquired infection with Syphacia obvelata in a number of mouse strains housed in a non-barrier facility. These mice, which included cross-bred and congenic, inbred strains on various genetic backgrounds, differ in the loci for the immune function genes--major histocompatibility complex class II (MHCII), toll-like receptor 4 (Tlr4), and solute carrier family 11, member 1 (Slc11a1)--which allowed comparisons of the impact of these genes on resistance to pinworm infection. Male and female mice of various ages were sampled over an 18-month period; infection was determined by use of the cellophane tape test. Results indicated that mice that were MHCII+/+ had a significantly lower prevalence of infection than did mice that were MHCII-/-. Differences were not seen between male and female mice. Although MHCII+/+ mice had an age-associated decrease in infection prevalence, such decrease was not seen in MHCII-/- mice. In contrast, infection prevalence in mice with the normal Tlr4 gene (Tlr4(LPS-n/LPS-n)) gene did not differ significantly compared with that in mice that were homozygous for either the point mutation (Tlr4(LPS-d/LPS-d)) or deletion (Tlr4(LPS-del/LPS-del)) of that gene. Likewise, the presence (Sle11a1r/r) or absence (Slc11a1s/s) of functional alleles for Slc11a1 had no effect on the prevalence of infection with S. obvelata. In conclusion, presence of MHCII, but not Tlr4 or Slc11a1 significantly influences prevalence of naturally acquired infection with S. obvelata. These data justify further comprehensive analyses of the immune components that are involved in pinworm resistance.

  15. Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

    PubMed Central

    Hazra, Sudipta; Glaser, Jan; Holzgrabe, Ulrike; Hazra, Banasri; Schurigt, Uta

    2015-01-01

    Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches. PMID:26554591

  16. Identification of Body Fat Mass as a Major Determinant of Metabolic Rate in Mice

    PubMed Central

    Kaiyala, Karl J.; Morton, Gregory J.; Leroux, Brian G.; Ogimoto, Kayoko; Wisse, Brent; Schwartz, Michael W.

    2010-01-01

    OBJECTIVE Analysis of energy expenditure (EE) in mice is essential to obesity research. Since EE varies with body mass, comparisons between lean and obese mice are confounded unless EE is normalized to account for body mass differences. We 1) assessed the validity of ratio-based EE normalization involving division of EE by either total body mass (TBM) or lean body mass (LBM), 2) compared the independent contributions of LBM and fat mass (FM) to EE, and 3) investigated whether leptin contributes to the link between FM and EE. RESEARCH DESIGN AND METHODS We used regression modeling of calorimetry and body composition data in 137 mice to estimate the independent contributions of LBM and FM to EE. Subcutaneous administration of leptin or vehicle to 28 obese ob/ob mice and 32 fasting wild-type mice was used to determine if FM affects EE via a leptin-dependent mechanism. RESULTS Division of EE by either TBM or LBM is confounded by body mass variation. The contribution of FM to EE is comparable to that of LBM in normal mice (expressed per gram of tissue) but is absent in leptin-deficient ob/ob mice. When leptin is administered at physiological doses, the plasma leptin concentration supplants FM as an independent determinant of EE in both ob/ob mice and normal mice rendered leptin-deficient by fasting. CONCLUSIONS The contribution of FM to EE is substantially greater than predicted from the metabolic cost of adipose tissue per se, and the mechanism underlying this effect is leptin dependent. Regression-based approaches that account for variation in both FM and LBM are recommended for normalization of EE in mice. PMID:20413511

  17. Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

    PubMed Central

    Guilloteau, Laurence A.; Dornand, Jacques; Gross, Antoine; Olivier, Michel; Cortade, Fabienne; Vern, Yves Le; Kerboeuf, Dominique

    2003-01-01

    Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene

  18. P23H opsin knock-in mice reveal a novel step in retinal rod disc morphogenesis

    PubMed Central

    Sakami, Sanae; Kolesnikov, Alexander V.; Kefalov, Vladimir J.; Palczewski, Krzysztof

    2014-01-01

    Retinal rod photoreceptor cells have double membrane discs located in their outer segments (ROS) that are continuously formed proximally from connecting cilia (CC) and phagocytized distally by the retinal pigmented epithelium. The major component of these rod discs, the light-sensitive visual pigment rhodopsin (Rho), consists of an opsin protein linked to 11-cis-retinal. The P23H mutation of rod opsin (P23H opsin) is the most common cause of human blinding autosomal dominant retinitis pigmentosa (adRP). A mouse model of adRP with this mutation (RhoP23H/+) shows low levels of P23H opsin protein, partial misalignment of discs and progressive retinal degeneration. However, the impact of mutant P23H opsin on the formation of abnormal discs is unclear and it is still unknown whether this mutant pigment can mediate phototransduction. Using transretinal ERG recordings, we demonstrate that P23H mutant Rho can trigger phototransduction but RhoP23H/P23H rods are ∼17 000-fold less sensitive to light than Rho+/+ rods and produce abnormally fast photo-responses. By analyzing homozygous RhoP23H/P23H knock-in mice, we show that P23H opsin is transported to ciliary protrusions where it forms sagittally elongated discs. Transmission electron microscopy of postnatal day (PND) 14 RhoP23H/+ mouse retina revealed disordered sagittally oriented discs before the onset of retinal degeneration. Surprisingly, we also observed smaller, immature sagittally oriented discs in PND14 Rho+/− and Rho+/+ mice that were not seen in older animals. These findings provide fundamental insights into the pathogenesis of the P23H mutant opsin and reveal a novel early sagittally aligned disc formation step in normal ROS disc expansion. PMID:24214395

  19. A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice.

    PubMed

    Wortmann, Markus; Hakimi, Maani; Fleming, Thomas; Peters, Andreas S; Sijmonsma, Tjeerd P; Herzig, Stephan; Nawroth, Peter P; Böckler, Dittmar; Dihlmann, Susanne

    2016-01-01

    Objective. Glyoxalase-1 is an enzyme detoxifying methylglyoxal (MG). MG is a potent precursor of advanced glycation endproducts which are regarded to be a key player in micro- and macrovascular damage. Yet, the role of Glo1 in atherosclerosis remains unclear. In this study, the effect of Glo1 on mouse metabolism and atherosclerosis is evaluated. Methods. Glo1 knockdown mice were fed a high fat or a standard diet for 10 weeks. Body weight and composition were investigated by Echo MRI. The PhenoMaster system was used to measure the energy expenditure. To evaluate the impact of Glo1 on atherosclerosis, Glo1(KD) mice were crossed with ApoE-knockout mice and fed a high fat diet for 14 weeks. Results. Glo1 activity was significantly reduced in heart, liver, and kidney lysates derived from Glo1(KD) mice. Yet, there was no increase in methylglyoxal-derived AGEs in all organs analyzed. The Glo1 knockdown did not affect body weight or body composition. Metabolic studies via indirect calorimetry did not show significant effects on energy expenditure. Glo1(KD) mice crossed to ApoE(-/-) mice did not show enhanced formation of atherosclerosis. Conclusion. A Glo1 knockdown does not have major short term effects on the energy expenditure or the formation of atherosclerotic plaques.

  20. Common and unique mechanisms of Chinese herbal remedies on ischemic stroke mice revealed by transcriptome analyses.

    PubMed

    Shen, Yuh-Chiang; Lu, Chung-Kuang; Liou, Kuo-Tong; Hou, Yu-Chang; Lin, Yun-Lan; Wang, Yea-Hwey; Sun, Hsing-Jen; Liao, Ko-Hsun; Wang, Hsei-Wei

    2015-09-15

    Four traditional Chinese herbal remedies (CHR) including Buyang Huanwu decoction (BHD), Xuefu Zhuyu decoction (XZD), Tianma Gouteng decoction (TGD) and Shengyu decoction (SYD) are popular used in treating brain-related dysfunction clinically with different syndrome/pattern based on traditional Chinese medicine (TCM) principles, yet their neuroprotective mechanisms are still unclear. Mice were subjected to an acute ischemic stroke to examine the efficacy and molecular mechanisms of action underlying these CHR. CHR treatment significantly enhanced the survival rate of stroke mice, with BHD being the most effective CHR. All CHR were superior to recombinant tissue-type plasminogen activator (rt-PA) treatment in successfully ameliorating brain function, infarction, and neurological deficits in stroke mice that also paralleled to improvements in blood-brain barrier damage, inflammation, apoptosis, and neurogenesis. Transcriptome analyses reveals that a total of 774 ischemia-induced probe sets were significantly modulated by four CHR, including 52 commonly upregulated genes and 54 commonly downregulated ones. Among them, activation of neurogenesis-associated signaling pathways and down-regulating inflammation and apoptosis pathways are key common mechanisms in ischemic stroke protection by all CHR. Besides, levels of plasma CX3CL1 and S100a9 in patients could be used as biomarkers for therapeutic evaluation before functional recovery could be observed. Our results suggest that using CHR, a combinatory cocktail therapy, is a better way than rt-PA for treating cerebral ischemic-associated diseases through modulating a common as well as a specific group of genes/pathways that may partially explain the syndrome differentiation and treatment principle in TCM. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Creatine Revealed Anticonvulsant Properties on Chemically and Electrically Induced Seizures in Mice.

    PubMed

    Shafaroodi, Hamed; Shahbek, Farnaz; Faizi, Mehrdad; Ebrahimi, Farzad; Moezi, Leila

    2016-01-01

    Creatine exerts beneficial effects on a variety of pathologies in which energy metabolism and oxidative stress play an etiological role. Creatine supplements have shown beneficial effects on neurological disorders including Parkinson׳s disease, Huntington›s disease, amyotrophic lateral sclerosis, as well as Alzheimer›s disease and stroke. However, the potential benefits of creatine for patients with convulsive disorders remain poorly defined. While some authors did not suggest any anti- or pro-convulsant roles for creatine treatment, others suggest that creatine may be an anticonvulsant agent. In this study, we investigated the effects of creatine on seizures in mice. Three models were used to explore the role of creatine on seizures in mice including intravenous pentylenetetrazole (PTZ), intraperitoneal PTZ, and electroshock models. Acute creatine treatment (10, 20, 40 and 80 mg/Kg) significantly increased the clonic seizure threshold in the intravenous PTZ model. Sub-chronic administration of creatine (10 and 20 mg/Kg) revealed a significant anticonvulsant effect in intravenous PTZ model. Acute creatine administration (10, 20 and 40 mg/Kg) significantly decreased the frequency of clonic seizures in the intraperitoneal PTZ model. Besides, acute creatine (40 and 80 mg/Kg) decreased the incidence of tonic seizures after electroshock. In conclusion, creatine exerts anticonvulsant effects in three seizure models; therefore, it may act as a potential drug to help patients with convulsions. However, further investigations should be done to clarify these results more.

  2. Creatine Revealed Anticonvulsant Properties on Chemically and Electrically Induced Seizures in Mice

    PubMed Central

    Shafaroodi, Hamed; Shahbek, Farnaz; Faizi, Mehrdad; Ebrahimi, Farzad; Moezi, Leila

    2016-01-01

    Creatine exerts beneficial effects on a variety of pathologies in which energy metabolism and oxidative stress play an etiological role. Creatine supplements have shown beneficial effects on neurological disorders including Parkinson׳s disease, Huntington›s disease, amyotrophic lateral sclerosis, as well as Alzheimer›s disease and stroke. However, the potential benefits of creatine for patients with convulsive disorders remain poorly defined. While some authors did not suggest any anti- or pro-convulsant roles for creatine treatment, others suggest that creatine may be an anticonvulsant agent. In this study, we investigated the effects of creatine on seizures in mice. Three models were used to explore the role of creatine on seizures in mice including intravenous pentylenetetrazole (PTZ), intraperitoneal PTZ, and electroshock models. Acute creatine treatment (10, 20, 40 and 80 mg/Kg) significantly increased the clonic seizure threshold in the intravenous PTZ model. Sub-chronic administration of creatine (10 and 20 mg/Kg) revealed a significant anticonvulsant effect in intravenous PTZ model. Acute creatine administration (10, 20 and 40 mg/Kg) significantly decreased the frequency of clonic seizures in the intraperitoneal PTZ model. Besides, acute creatine (40 and 80 mg/Kg) decreased the incidence of tonic seizures after electroshock. In conclusion, creatine exerts anticonvulsant effects in three seizure models; therefore, it may act as a potential drug to help patients with convulsions. However, further investigations should be done to clarify these results more. PMID:28243281

  3. Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    2002-09-24

    Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

  4. Proteomic analysis of mice fed methionine and choline deficient diet reveals marker proteins associated with steatohepatitis.

    PubMed

    Lee, Su Jin; Kang, Jeong Han; Iqbal, Waqas; Kwon, Oh-Shin

    2015-01-01

    The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.

  5. Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice

    PubMed Central

    Zhang, Limin; Hatzakis, Emmanuel; Nichols, Robert G.; Hao, Ruixin; Correll, Jared; Smith, Philip B.; Chiaro, Christopher R.; Perdew, Gary H.; Patterson, Andrew D.

    2016-01-01

    Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) were assessed using global 1H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolic profiling of extracts obtained from serum and liver. 1H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was also observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure. PMID:26023891

  6. Targeted Deletion of p73 in Mice Reveals Its Role in T Cell Development and Lymphomagenesis

    PubMed Central

    Nemajerova, Alice; Palacios, Gustavo; Nowak, Norma J.; Matsui, Sei-ichi; Petrenko, Oleksi

    2009-01-01

    Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia. PMID:19907659

  7. Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis.

    PubMed

    Nemajerova, Alice; Palacios, Gustavo; Nowak, Norma J; Matsui, Sei-Ichi; Petrenko, Oleksi

    2009-11-11

    Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.

  8. Moesin-deficient mice reveal a non-redundant role for moesin in lymphocyte homeostasis.

    PubMed

    Hirata, Takako; Nomachi, Akira; Tohya, Kazuo; Miyasaka, Masayuki; Tsukita, Sachiko; Watanabe, Takeshi; Narumiya, Shuh

    2012-11-01

    Moesin is a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins. These proteins organize membrane domains by interacting with plasma membrane proteins and the actin cytoskeleton. Because of their high sequence similarity, ERM proteins are usually thought to be functionally redundant. Lymphocytes express two ERM proteins, ezrin and moesin. Whether each ERM plays a specialized role in lymphocytes, particularly in vivo, remains unknown. Here, we show that moesin has a crucial, non-redundant role in lymphocyte homeostasis. Moesin-deficient mice exhibited decreases in both T and B cells in the peripheral blood and lymph nodes, but not in the spleen. This phenotype was recapitulated in bone marrow (BM) chimeras with a hematopoietic moesin deficiency. Although the T and B cells apparently developed without major defects in the moesin-deficient mice, T cell egress from the thymus and immature B cell egress from the BM were impaired. In the periphery, both T and B cells showed delayed egress from lymphoid organs. We showed that moesin is the primary phosphorylated ERM subject to dynamic regulation during cell shape changes and migration. Our findings identify a previously unknown, non-redundant function of moesin in lymphocyte homeostasis in regulating lymphocyte egress from lymphoid organs.

  9. Myelin/oligodendrocyte glycoprotein–deficient (MOG-deficient) mice reveal lack of immune tolerance to MOG in wild-type mice

    PubMed Central

    Delarasse, Cécile; Daubas, Philippe; Mars, Lennart T.; Vizler, Csaba; Litzenburger, Tobias; Iglesias, Antonio; Bauer, Jan; Della Gaspera, Bruno; Schubart, Anna; Decker, Laurence; Dimitri, Dalia; Roussel, Guy; Dierich, Andrée; Amor, Sandra; Dautigny, André; Liblau, Roland; Pham-Dinh, Danielle

    2003-01-01

    We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35–55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG–/– mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE. PMID:12925695

  10. H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

    SciTech Connect

    Blackwell, J.M.; Hale, C.; Roberts, M.B.; Ulczak, O.M.; Liew, F.Y.; Howard, J.G.

    1985-01-01

    The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncure phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.

  11. Behavioral characterization in a comprehensive mouse test battery reveals motor and sensory impairments in growth-associated protein-43 null mutant mice.

    PubMed

    Metz, G A; Schwab, M E

    2004-01-01

    The growth-associated protein (GAP)-43 is a major neuronal protein associated with axonal growth, neuronal plasticity and learning. The observation that only 5-10% of mice with a full GAP-43 gene deletion survive weaning suggests that basic neural functions are disturbed. Here we used a comprehensive test battery to characterise and quantify the motor and sensory function of surviving adult homozygous GAP-43 (-/-) mice as compared with GAP-43 (+/-) and wild-type animals. The test battery was comprised of motor, sensory, and reflex tests producing 25 measures of locomotion, as well as epicritic, auditory, olfactory and visual function. The analysis revealed significant impairments in muscle strength, limb coordination and balance in GAP-43 (-/-) mice. Furthermore, GAP-43 (-/-) animals were hyperactive and showed reduced anxiety as measured by open field and light dark tests. In sensory tests, GAP-43 (-/-) mice were tested for impaired tactile and labyrinthine function. Abnormal reflexes were found in the contact and vibrissa placing responses, and in the crossed extensor reflex. GAP-43 (+/-) animals showed only moderate abnormalities as compared with wild-type animals. We conclude that GAP-43 is necessary for the development and function of a variety of neuronal systems. The results also show that the comprehensive test battery used in the present study represents a sensitive approach to assess the functional integrity of ascending and descending pathways in genetically manipulated mice.

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

    PubMed

    Nakamura, Toru; Sato, Asako; Kitsukawa, Takashi; Momiyama, Toshihiko; Yamamori, Tetsuo; Sasaoka, Toshikuni

    2014-01-01

    Both D1R and D2R knock out (KO) mice of the major dopamine receptors show significant motor impairments. However, there are some discrepant reports, which may be due to the differences in genetic background and experimental procedures. In addition, only few studies directly compared the motor performance of D1R and D2R KO mice. In this paper, we examined the behavioral difference among N10 congenic D1R and D2R KO, and wild type (WT) mice. First, we examined spontaneous motor activity in the home cage environment for consecutive 5 days. Second, we examined motor performance using the rota-rod task, a standard motor task in rodents. Third, we examined motor ability with the Step-Wheel task in which mice were trained to run in a motor-driven turning wheel adjusting their steps on foothold pegs to drink water. The results showed clear differences among the mice of three genotypes in three different types of behavior. In monitoring spontaneous motor activities, D1R and D2R KO mice showed higher and lower 24 h activities, respectively, than WT mice. In the rota-rod tasks, at a low speed, D1R KO mice showed poor performance but later improved, whereas D2R KO mice showed a good performance at early days without further improvement. When first subjected to a high speed task, the D2R KO mice showed poorer rota-rod performance at a low speed than the D1R KO mice. In the Step-Wheel task, across daily sessions, D2R KO mice increased the duration that mice run sufficiently close to the spout to drink water, and decreased time to touch the floor due to missing the peg steps and number of times the wheel was stopped, which performance was much better than that of D1R KO mice. These incongruent results between the two tasks for D1R and D2R KO mice may be due to the differences in the motivation for the rota-rod and Step-Wheel tasks, aversion- and reward-driven, respectively. The Step-Wheel system may become a useful tool for assessing the motor ability of WT and mutant mice.

  13. Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization.

    PubMed

    Wu, Ângela C A; Freitas, Michelle A R; Silva, Soraia de O; Nogueira, Paula M; Soares, Rodrigo P; Pesquero, João Bosco; Gomes, Maria A; Pesquero, Jorge L; Melo, Maria N

    2015-01-01

    Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

  14. Isolation of glia from Alzheimer's mice reveals inflammation and dysfunction.

    PubMed

    Orre, Marie; Kamphuis, Willem; Osborn, Lana M; Jansen, Anne H P; Kooijman, Lieneke; Bossers, Koen; Hol, Elly M

    2014-12-01

    Reactive astrocytes and microglia are associated with amyloid plaques in Alzheimer's disease (AD). Yet, not much is known about the molecular alterations underlying this reactive phenotype. To get an insight into the molecular changes underlying AD induced astrocyte and microglia reactivity, we performed a transcriptional analysis on acutely isolated astrocytes and microglia from the cortex of aged controls and APPswe/PS1dE9 AD mice. As expected, both cell types acquired a proinflammatory phenotype, which confirms the validity of our approach. Interestingly, we observed that the immune alteration in astrocytes was relatively more pronounced than in microglia. Concurrently, our data reveal that astrocytes display a reduced expression of neuronal support genes and genes involved in neuronal communication. The microglia showed a reduced expression of phagocytosis and/or endocytosis genes. Co-expression analysis of a human AD expression data set and the astrocyte and microglia data sets revealed that the inflammatory changes in astrocytes were remarkably comparable in mouse and human AD, whereas the microglia changes showed less similarity. Based on these findings we argue that chronically proinflammatory astrocyte and microglia phenotypes, showing a reduction of genes involved in neuronal support and neuronal signaling, are likely to contribute to the neuronal dysfunction and cognitive decline in AD. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Genetic assignment of large seizures of elephant ivory reveals Africa’s major poaching hotspots

    PubMed Central

    Wasser, S. K.; Brown, L.; Mailand, C.; Mondol, S.; Clark, W.; Laurie, C.; Weir, B. S.

    2017-01-01

    Poaching of elephants is now occurring at rates that threaten African populations with extinction. Identifying the number and location of Africa’s major poaching hotspots may assist efforts to end poaching and facilitate recovery of elephant populations. We genetically assign origin to 28 large ivory seizures (≥0.5 metric tons) made between 1996 and 2014, also testing assignment accuracy. Results suggest that the major poaching hotspots in Africa may be currently concentrated in as few as two areas. Increasing law enforcement in these two hotspots could help curtail future elephant losses across Africa and disrupt this organized transnational crime. PMID:26089357

  16. CONSERVATION. Genetic assignment of large seizures of elephant ivory reveals Africa's major poaching hotspots.

    PubMed

    Wasser, S K; Brown, L; Mailand, C; Mondol, S; Clark, W; Laurie, C; Weir, B S

    2015-07-03

    Poaching of elephants is now occurring at rates that threaten African populations with extinction. Identifying the number and location of Africa's major poaching hotspots may assist efforts to end poaching and facilitate recovery of elephant populations. We genetically assign origin to 28 large ivory seizures (≥0.5 metric tons) made between 1996 and 2014, also testing assignment accuracy. Results suggest that the major poaching hotspots in Africa may be currently concentrated in as few as two areas. Increasing law enforcement in these two hotspots could help curtail future elephant losses across Africa and disrupt this organized transnational crime. Copyright © 2015, American Association for the Advancement of Science.

  17. Medial prefrontal aberrations in major depressive disorder revealed by cytoarchitectonically informed voxel-based morphometry

    PubMed Central

    Bludau, Sebastian; Bzdok, Danilo; Gruber, Oliver; Kohn, Nils; Riedl, Valentin; Sorg, Christian; Palomero-Gallagher, Nicola; Müller, Veronika I.; Hoffstaedter, Felix; Amunts, Katrin; Eickhoff, Simon B.

    2017-01-01

    Objective The heterogeneous human frontal pole has been identified as a node in the dysfunctional network of major depressive disorder. The contribution of the medial (socio-affective) versus lateral (cognitive) frontal pole to major depression pathogenesis is currently unclear. The present study performs morphometric comparison of the microstructurally informed subdivisions of human frontal pole between depressed patients and controls using both uni- and multivariate statistics. Methods Multi-site voxel- and region-based morphometric MRI analysis of 73 depressed patients and 73 matched controls without psychiatric history. Frontal pole volume was first compared between depressed patients and controls by subdivision-wise classical morphometric analysis. In a second approach, frontal pole volume was compared by subdivision-naive multivariate searchlight analysis based on support vector machines. Results Subdivision-wise morphometric analysis found a significantly smaller medial frontal pole in depressed patients with a negative correlation of disease severity and duration. Histologically uninformed multivariate voxel-wise statistics provided converging evidence for structural aberrations specific to the microstructurally defined medial area of the frontal pole in depressed patients. Conclusions Across disparate methods, we demonstrated subregion specificity in the left medial frontal pole volume in depressed patients. Indeed, the frontal pole was shown to structurally and functionally connect to other key regions in major depression pathology like the anterior cingulate cortex and the amygdala via the uncinate fasciculus. Present and previous findings consolidate the left medial portion of the frontal pole as particularly altered in major depression. PMID:26621569

  18. Immunization with major outer membrane proteins in experimental salmonellosis of mice.

    PubMed Central

    Kuusi, N; Nurminen, M; Saxen, H; Valtonen, M; Mäkelä, P H

    1979-01-01

    Porin (outer membrane protein) preparations extracted from a rough (Rb2) mutant of Salmonella typhimurium proved to be good immunogens in mice and rabbits. The antibody response achieved was measured by using enzyme-linked immunosorbent assay techniques. High titers of both antiporin and antilipopolysaccharide were detected in both species. The rabbit antiserum raised against the porins and the porin preparations themselves had a highly significant protective capacity against intraperitoneal Salmonella infection of mice. Absorption of the rabbit antiporin serum with lipopolysaccharide immunosorbent did not change its protective capacity in a passive immunization experiment, suggesting that the antiporin antibody preparations were the active components. Images PMID:387596

  19. Evolution of Modern Birds Revealed by Mitogenomics: Timing the Radiation and Origin of Major Orders

    PubMed Central

    Pacheco, M. Andreína; Battistuzzi, Fabia U.; Lentino, Miguel; Aguilar, Roberto F.; Kumar, Sudhir; Escalante, Ananias A.

    2011-01-01

    Mitochondrial (mt) genes and genomes are among the major sources of data for evolutionary studies in birds. This places mitogenomic studies in birds at the core of intense debates in avian evolutionary biology. Indeed, complete mt genomes are actively been used to unveil the phylogenetic relationships among major orders, whereas single genes (e.g., cytochrome c oxidase I [COX1]) are considered standard for species identification and defining species boundaries (DNA barcoding). In this investigation, we study the time of origin and evolutionary relationships among Neoaves orders using complete mt genomes. First, we were able to solve polytomies previously observed at the deep nodes of the Neoaves phylogeny by analyzing 80 mt genomes, including 17 new sequences reported in this investigation. As an example, we found evidence indicating that columbiforms and charadriforms are sister groups. Overall, our analyses indicate that by improving the taxonomic sampling, complete mt genomes can solve the evolutionary relationships among major bird groups. Second, we used our phylogenetic hypotheses to estimate the time of origin of major avian orders as a way to test if their diversification took place prior to the Cretaceous/Tertiary (K/T) boundary. Such timetrees were estimated using several molecular dating approaches and conservative calibration points. Whereas we found time estimates slightly younger than those reported by others, most of the major orders originated prior to the K/T boundary. Finally, we used our timetrees to estimate the rate of evolution of each mt gene. We found great variation on the mutation rates among mt genes and within different bird groups. COX1 was the gene with less variation among Neoaves orders and the one with the least amount of rate heterogeneity across lineages. Such findings support the choice of COX 1 among mt genes as target for developing DNA barcoding approaches in birds. PMID:21242529

  20. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

    PubMed Central

    Smyllie, Nicola J.; Chesham, Johanna E.; Hamnett, Ryan; Maywood, Elizabeth S.; Hastings, Michael H.

    2016-01-01

    The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. PMID:26966234

  1. Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III).

    PubMed

    Kowalewski, Björn; Heimann, Peter; Ortkras, Theresa; Lüllmann-Rauch, Renate; Sawada, Tomo; Walkley, Steven U; Dierks, Thomas; Damme, Markus

    2015-04-01

    Deficiency of arylsulfatase G (ARSG) leads to a lysosomal storage disease in mice resembling biochemical and pathological features of the mucopolysaccharidoses and particularly features of mucopolysaccharidosis type III (Sanfilippo syndrome). Here we show that Arsg KO mice share common neuropathological findings with other Sanfilippo syndrome models and patients, but they can be clearly distinguished by the limitation of most phenotypic alterations to the cerebellum, presenting with ataxia as the major neurological finding. We determined in detail the expression of ARSG in the central nervous system and observed highest expression in perivascular macrophages (which are characterized by abundant vacuolization in Arsg KO mice) and oligodendrocytes. To gain insight into possible mechanisms leading to ataxia, the pathology in older adult mice (>12 months) was investigated in detail. This study revealed massive loss of Purkinje cells and gliosis in the cerebellum, and secondary accumulation of glycolipids like GM2 and GM3 gangliosides and unesterified cholesterol in surviving Purkinje cells, as well as neurons of some other brain regions. The abundant presence of ubiquitin and p62-positive aggregates in degenerating Purkinje cells coupled with the absence of significant defects in macroautophagy is consistent with lysosomal membrane permeabilization playing a role in the pathogenesis of Arsg-deficient mice and presumably Sanfilippo disease in general. Our data delineating the phenotype of mucopolysaccharidosis IIIE in a mouse KO model should help in the identification of possible human cases of this disease. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Recovery from Friend disease in mice with reduced major histocompatibility complex class I expression.

    PubMed Central

    Hasenkrug, K J; Sprangrude, G J; Nishio, J; Brooks, D M; Chesebro, B

    1994-01-01

    Mice homozygous for the b allele of the MHC gene, H-2D, have a high incidence of recovery from Friend virus infections, while mice heterozygous for the b allele at H-2D have a very low incidence of recovery. Previous experiments indicated that the low recovery rates associated with heterozygosity at H-2D might be related to a gene dosage effect requiring the expression of two H-2Db alleles for high recovery. We investigated the effects of reduced H-2Db expression on recovery from Friend disease by using H-2b homozygous mice carrying a single beta 2-microglobulin gene disruption. These mice had reductions in cell surface H-2Db expression comparable to those of H-2Da/b heterozygotes. Numerous cell types with various levels of H-2Db expression were examined, and in each case, the expression levels in the beta 2-microglobulin mutants closely reflected those observed in the H-2Da/b heterozygotes. We found, however, that reduced expression did not affect recovery from Friend disease, indicating that heterozygous levels of H-2Db expression are sufficient for the high-recovery phenotype previously associated only with H-2Db homozygotes. PMID:8138991

  3. Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice.

    PubMed

    Gallego, Sandra F; Sprenger, Richard R; Neess, Ditte; Pauling, Josch K; Færgeman, Nils J; Ejsing, Christer S

    2017-02-01

    The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.

  4. 3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin.

    PubMed

    Booth, Daniel G; Beckett, Alison J; Molina, Oscar; Samejima, Itaru; Masumoto, Hiroshi; Kouprina, Natalay; Larionov, Vladimir; Prior, Ian A; Earnshaw, William C

    2016-11-17

    Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Dynamic functional connectivity reveals altered variability in functional connectivity among patients with major depressive disorder.

    PubMed

    Demirtaş, Murat; Tornador, Cristian; Falcón, Carles; López-Solà, Marina; Hernández-Ribas, Rosa; Pujol, Jesús; Menchón, José M; Ritter, Petra; Cardoner, Narcis; Soriano-Mas, Carles; Deco, Gustavo

    2016-08-01

    Resting-state fMRI (RS-fMRI) has become a useful tool to investigate the connectivity structure of mental health disorders. In the case of major depressive disorder (MDD), recent studies regarding the RS-fMRI have found abnormal connectivity in several regions of the brain, particularly in the default mode network (DMN). Thus, the relevance of the DMN to self-referential thoughts and ruminations has made the use of the resting-state approach particularly important for MDD. The majority of such research has relied on the grand averaged functional connectivity measures based on the temporal correlations between the BOLD time series of various brain regions. We, in our study, investigated the variations in the functional connectivity over time at global and local level using RS-fMRI BOLD time series of 27 MDD patients and 27 healthy control subjects. We found that global synchronization and temporal stability were significantly increased in the MDD patients. Furthermore, the participants with MDD showed significantly increased overall average (static) functional connectivity (sFC) but decreased variability of functional connectivity (vFC) within specific networks. Static FC increased to predominance among the regions pertaining to the default mode network (DMN), while the decreased variability of FC was observed in the connections between the DMN and the frontoparietal network. Hum Brain Mapp 37:2918-2930, 2016. © 2016 Wiley Periodicals, Inc.

  6. Dynamic functional connectivity reveals altered variability in functional connectivity among patients with major depressive disorder

    PubMed Central

    Tornador, Cristian; Falcón, Carles; López‐Solà, Marina; Hernández‐Ribas, Rosa; Pujol, Jesús; Menchón, José M.; Ritter, Petra; Cardoner, Narcis; Soriano‐Mas, Carles; Deco, Gustavo

    2016-01-01

    Abstract Resting‐state fMRI (RS‐fMRI) has become a useful tool to investigate the connectivity structure of mental health disorders. In the case of major depressive disorder (MDD), recent studies regarding the RS‐fMRI have found abnormal connectivity in several regions of the brain, particularly in the default mode network (DMN). Thus, the relevance of the DMN to self‐referential thoughts and ruminations has made the use of the resting‐state approach particularly important for MDD. The majority of such research has relied on the grand averaged functional connectivity measures based on the temporal correlations between the BOLD time series of various brain regions. We, in our study, investigated the variations in the functional connectivity over time at global and local level using RS‐fMRI BOLD time series of 27 MDD patients and 27 healthy control subjects. We found that global synchronization and temporal stability were significantly increased in the MDD patients. Furthermore, the participants with MDD showed significantly increased overall average (static) functional connectivity (sFC) but decreased variability of functional connectivity (vFC) within specific networks. Static FC increased to predominance among the regions pertaining to the default mode network (DMN), while the decreased variability of FC was observed in the connections between the DMN and the frontoparietal network. Hum Brain Mapp 37:2918–2930, 2016. © 2016 Wiley Periodicals, Inc. PMID:27120982

  7. Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice.

    PubMed

    Pascolo, Lorella; Zabucchi, Giuliano; Gianoncelli, Alessandra; Kourousias, George; Trevisan, Elisa; Pascotto, Ernesto; Casarsa, Claudia; Ryan, Chris; Lucattelli, Monica; Lungarella, Giuseppe; Cavarra, Eleonora; Bartalesi, Barbara; Zweyer, Marina; Cammisuli, Francesca; Melato, Mauro; Borelli, Violetta

    2016-01-22

    Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity.

    PubMed

    Everard, Amandine; Lazarevic, Vladimir; Gaïa, Nadia; Johansson, Maria; Ståhlman, Marcus; Backhed, Fredrik; Delzenne, Nathalie M; Schrenzel, Jacques; François, Patrice; Cani, Patrice D

    2014-10-01

    The gut microbiota is involved in metabolic and immune disorders associated with obesity and type 2 diabetes. We previously demonstrated that prebiotic treatment may significantly improve host health by modulating bacterial species related to the improvement of gut endocrine, barrier and immune functions. An analysis of the gut metagenome is needed to determine which bacterial functions and taxa are responsible for beneficial microbiota-host interactions upon nutritional intervention. We subjected mice to prebiotic (Pre) treatment under physiological (control diet: CT) and pathological conditions (high-fat diet: HFD) for 8 weeks and investigated the production of intestinal antimicrobial peptides and the gut microbiome. HFD feeding significantly decreased the expression of regenerating islet-derived 3-gamma (Reg3g) and phospholipase A2 group-II (PLA2g2) in the jejunum. Prebiotic treatment increased Reg3g expression (by ∼50-fold) and improved intestinal homeostasis as suggested by the increase in the expression of intectin, a key protein involved in intestinal epithelial cell turnover. Deep metagenomic sequencing analysis revealed that HFD and prebiotic treatment significantly affected the gut microbiome at different taxonomic levels. Functional analyses based on the occurrence of clusters of orthologous groups (COGs) of proteins also revealed distinct profiles for the HFD, Pre, HFD-Pre and CT groups. Finally, the gut microbiota modulations induced by prebiotics counteracted HFD-induced inflammation and related metabolic disorders. Thus, we identified novel putative taxa and metabolic functions that may contribute to the development of or protection against the metabolic alterations observed during HFD feeding and HFD-Pre feeding.

  9. Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity

    PubMed Central

    Everard, Amandine; Lazarevic, Vladimir; Gaïa, Nadia; Johansson, Maria; Ståhlman, Marcus; Backhed, Fredrik; Delzenne, Nathalie M; Schrenzel, Jacques; François, Patrice; Cani, Patrice D

    2014-01-01

    The gut microbiota is involved in metabolic and immune disorders associated with obesity and type 2 diabetes. We previously demonstrated that prebiotic treatment may significantly improve host health by modulating bacterial species related to the improvement of gut endocrine, barrier and immune functions. An analysis of the gut metagenome is needed to determine which bacterial functions and taxa are responsible for beneficial microbiota–host interactions upon nutritional intervention. We subjected mice to prebiotic (Pre) treatment under physiological (control diet: CT) and pathological conditions (high-fat diet: HFD) for 8 weeks and investigated the production of intestinal antimicrobial peptides and the gut microbiome. HFD feeding significantly decreased the expression of regenerating islet-derived 3-gamma (Reg3g) and phospholipase A2 group-II (PLA2g2) in the jejunum. Prebiotic treatment increased Reg3g expression (by ∼50-fold) and improved intestinal homeostasis as suggested by the increase in the expression of intectin, a key protein involved in intestinal epithelial cell turnover. Deep metagenomic sequencing analysis revealed that HFD and prebiotic treatment significantly affected the gut microbiome at different taxonomic levels. Functional analyses based on the occurrence of clusters of orthologous groups (COGs) of proteins also revealed distinct profiles for the HFD, Pre, HFD-Pre and CT groups. Finally, the gut microbiota modulations induced by prebiotics counteracted HFD-induced inflammation and related metabolic disorders. Thus, we identified novel putative taxa and metabolic functions that may contribute to the development of or protection against the metabolic alterations observed during HFD feeding and HFD-Pre feeding. PMID:24694712

  10. Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

    PubMed Central

    Calcagnì, Alessia; kors, Lotte; Verschuren, Eric; De Cegli, Rossella; Zampelli, Nicolina; Nusco, Edoardo; Confalonieri, Stefano; Bertalot, Giovanni; Pece, Salvatore; Settembre, Carmine; Malouf, Gabriel G; Leemans, Jaklien C; de Heer, Emile; Salvatore, Marco; Peters, Dorien JM; Di Fiore, Pier Paolo; Ballabio, Andrea

    2016-01-01

    TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway. DOI: http://dx.doi.org/10.7554/eLife.17047.001 PMID:27668431

  11. Jagged 1 is a major Notch ligand along cholangiocarcinoma development in mice and humans

    PubMed Central

    Che, L; Fan, B; Pilo, M G; Xu, Z; Liu, Y; Cigliano, A; Cossu, A; Palmieri, G; Pascale, R M; Porcu, A; Vidili, G; Serra, M; Dombrowski, F; Ribback, S; Calvisi, D F; Chen, X

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly malignancy with limited treatment options. Activation of the Notch signalling cascade has been implicated in cholangiocarcinogenesis. However, while several studies focused on the Notch receptors required for ICC development, little is known about the upstream inducers responsible for their activation. Here, we show that the Jagged 1 (Jag1) ligand is almost ubiquitously upregulated in human ICC samples when compared with corresponding non-tumorous counterparts. Furthermore, we found that while overexpression of Jag1 alone does not lead to liver tumour development, overexpression of Jag1 synergizes with activated AKT signalling to promote liver carcinogenesis in AKT/Jag1 mice. Histologically, tumours consisted exclusively of ICC, with hepatocellular tumours not occurring in AKT/Jag1 mice. Furthermore, tumours from AKT/Jag1 mice exhibited extensive desmoplastic reaction, an important feature of human ICC. At the molecular level, we found that both AKT/mTOR and Notch cascades are activated in AKT/Jag1 ICC tissues, and that the Notch signalling is necessary for ICC development in AKT/Jag1 mice. In human ICC cell lines, silencing of Jag1 via specific small interfering RNA reduces proliferation and increases apoptosis. Finally, combined inhibition of AKT and Notch pathways is highly detrimental for the in vitro growth of ICC cell lines. In summary, our study demonstrates that Jag1 is an important upstream inducer of the Notch signalling in human and mouse ICC. Targeting Jag1 might represent a novel therapeutic strategy for the treatment of this deadly disease. PMID:27918553

  12. Genetic dissection of pheromone processing reveals main olfactory system-mediated social behaviors in mice.

    PubMed

    Matsuo, Tomohiko; Hattori, Tatsuya; Asaba, Akari; Inoue, Naokazu; Kanomata, Nobuhiro; Kikusui, Takefumi; Kobayakawa, Reiko; Kobayakawa, Ko

    2015-01-20

    Most mammals have two major olfactory subsystems: the main olfactory system (MOS) and vomeronasal system (VNS). It is now widely accepted that the range of pheromones that control social behaviors are processed by both the VNS and the MOS. However, the functional contributions of each subsystem in social behavior remain unclear. To genetically dissociate the MOS and VNS functions, we established two conditional knockout mouse lines that led to either loss-of-function in the entire MOS or in the dorsal MOS. Mice with whole-MOS loss-of-function displayed severe defects in active sniffing and poor survival through the neonatal period. In contrast, when loss-of-function was confined to the dorsal MOB, sniffing behavior, pheromone recognition, and VNS activity were maintained. However, defects in a wide spectrum of social behaviors were observed: attraction to female urine and the accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and risk-assessment behaviors in response to an alarm pheromone. Functional dissociation of pheromone detection and pheromonal induction of behaviors showed the anterior olfactory nucleus (AON)-regulated social behaviors downstream from the MOS. Lesion analysis and neural activation mapping showed pheromonal activation in multiple amygdaloid and hypothalamic nuclei, important regions for the expression of social behavior, was dependent on MOS and AON functions. Identification of the MOS-AON-mediated pheromone pathway may provide insights into pheromone signaling in animals that do not possess a functional VNS, including humans.

  13. Genetic dissection of pheromone processing reveals main olfactory system-mediated social behaviors in mice

    PubMed Central

    Matsuo, Tomohiko; Hattori, Tatsuya; Asaba, Akari; Inoue, Naokazu; Kanomata, Nobuhiro; Kikusui, Takefumi; Kobayakawa, Reiko; Kobayakawa, Ko

    2015-01-01

    Most mammals have two major olfactory subsystems: the main olfactory system (MOS) and vomeronasal system (VNS). It is now widely accepted that the range of pheromones that control social behaviors are processed by both the VNS and the MOS. However, the functional contributions of each subsystem in social behavior remain unclear. To genetically dissociate the MOS and VNS functions, we established two conditional knockout mouse lines that led to either loss-of-function in the entire MOS or in the dorsal MOS. Mice with whole-MOS loss-of-function displayed severe defects in active sniffing and poor survival through the neonatal period. In contrast, when loss-of-function was confined to the dorsal MOB, sniffing behavior, pheromone recognition, and VNS activity were maintained. However, defects in a wide spectrum of social behaviors were observed: attraction to female urine and the accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and risk-assessment behaviors in response to an alarm pheromone. Functional dissociation of pheromone detection and pheromonal induction of behaviors showed the anterior olfactory nucleus (AON)-regulated social behaviors downstream from the MOS. Lesion analysis and neural activation mapping showed pheromonal activation in multiple amygdaloid and hypothalamic nuclei, important regions for the expression of social behavior, was dependent on MOS and AON functions. Identification of the MOS-AON–mediated pheromone pathway may provide insights into pheromone signaling in animals that do not possess a functional VNS, including humans. PMID:25564662

  14. PKN3 is the major regulator of angiogenesis and tumor metastasis in mice

    PubMed Central

    Mukai, Hideyuki; Muramatsu, Aiko; Mashud, Rana; Kubouchi, Koji; Tsujimoto, Sho; Hongu, Tsunaki; Kanaho, Yasunori; Tsubaki, Masanobu; Nishida, Shozo; Shioi, Go; Danno, Sally; Mehruba, Mona; Satoh, Ryosuke; Sugiura, Reiko

    2016-01-01

    PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin β1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes. PMID:26742562

  15. Structure of an endogenous yeast 26S proteasome reveals two major conformational states

    PubMed Central

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J.; Shi, Yigong; Wang, Feng

    2016-01-01

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function. PMID:26929360

  16. Structure of an endogenous yeast 26S proteasome reveals two major conformational states.

    PubMed

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J; Shi, Yigong; Wang, Feng

    2016-03-08

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.

  17. Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer

    PubMed Central

    Mine, Karina L.; Shulzhenko, Natalia; Yambartsev, Anatoly; Rochman, Mark; Sanson, Gerdine F. O.; Lando, Malin; Varma, Sudhir; Skinner, Jeff; Volfovsky, Natalia; Deng, Tao; Brenna, Sylvia M. F.; Carvalho, Carmen R. N.; Ribalta, Julisa C. L.; Bustin, Michael; Matzinger, Polly; Silva, Ismael D. C. G.; Lyng, Heidi; Gerbase-DeLima, Maria; Morgun, Andrey

    2014-01-01

    Although human papillomavirus (HPV) was identified as an etiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major sub-networks up-regulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal HPV decline in women with invasive cancer who were previously unable to clear the virus. PMID:23651994

  18. Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer.

    PubMed

    Mine, Karina L; Shulzhenko, Natalia; Yambartsev, Anatoly; Rochman, Mark; Sanson, Gerdine F O; Lando, Malin; Varma, Sudhir; Skinner, Jeff; Volfovsky, Natalia; Deng, Tao; Brenna, Sylvia M F; Carvalho, Carmen R N; Ribalta, Julisa C L; Bustin, Michael; Matzinger, Polly; Silva, Ismael D C G; Lyng, Heidi; Gerbase-DeLima, Maria; Morgun, Andrey

    2013-01-01

    Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.

  19. Knockout of major leaf ferredoxin reveals new redox-regulatory adaptations in Arabidopsis thaliana.

    PubMed

    Voss, Ingo; Koelmann, Meike; Wojtera, Joanna; Holtgrefe, Simone; Kitzmann, Camillo; Backhausen, Jan E; Scheibe, Renate

    2008-07-01

    Ferredoxins are the major distributors for electrons to the various acceptor systems in plastids. In green tissues, ferredoxins are reduced by photosynthetic electron flow in the light, while in heterotrophic tissues, nicotinamide adenine dinucleotide (reduced) (NADPH) generated in the oxidative pentose-phosphate pathway (OPP) is the reductant. We have used a Ds-T-DNA insertion line of Arabidopsis thaliana for the gene encoding the major leaf ferredoxin (Fd2, At1g60950) to create a situation of high electron pressure in the thylakoids. Although these plants (Fd2-KO) possess only the minor fraction of leaf Fd1 (At1g10960), they grow photoautotrophically on soil, but with a lower growth rate and less chlorophyll. The more oxidized conditions in the stroma due to the formation of reactive oxygen species are causing a re-adjustment of the redox state in these plants that helps them to survive even under high light. Redox homeostasis is achieved by regulation at both, the post-translational and the transcriptional level. Over-reduction of the electron transport chain leads to increased transcription of the malate-valve enzyme NADP-malate dehydrogenase (MDH), and the oxidized stroma leads to an increased transcription of the OPP enzyme glucose-6-P dehydrogenase. In isolated spinach chloroplasts, oxidized conditions give rise to a decreased activation state of NADP-MDH and an activation of glucose-6-P dehydrogenase even in the light. In Fd2-KO plants, NADPH-requiring antioxidant systems are upregulated. These adjustments must be caused by plastid signals, and they prevent oxidative damage under rather severe conditions.

  20. Major Soybean Maturity Gene Haplotypes Revealed by SNPViz Analysis of 72 Sequenced Soybean Genomes

    PubMed Central

    Langewisch, Tiffany; Zhang, Hongxin; Vincent, Ryan; Joshi, Trupti; Xu, Dong; Bilyeu, Kristin

    2014-01-01

    In this Genomics Era, vast amounts of next-generation sequencing data have become publicly available for multiple genomes across hundreds of species. Analyses of these large-scale datasets can become cumbersome, especially when comparing nucleotide polymorphisms across many samples within a dataset and among different datasets or organisms. To facilitate the exploration of allelic variation and diversity, we have developed and deployed an in-house computer software to categorize and visualize these haplotypes. The SNPViz software enables users to analyze region-specific haplotypes from single nucleotide polymorphism (SNP) datasets for different sequenced genomes. The examination of allelic variation and diversity of important soybean [Glycine max (L.) Merr.] flowering time and maturity genes may provide additional insight into flowering time regulation and enhance researchers' ability to target soybean breeding for particular environments. For this study, we utilized two available soybean genomic datasets for a total of 72 soybean genotypes encompassing cultivars, landraces, and the wild species Glycine soja. The major soybean maturity genes E1, E2, E3, and E4 along with the Dt1 gene for plant growth architecture were analyzed in an effort to determine the number of major haplotypes for each gene, to evaluate the consistency of the haplotypes with characterized variant alleles, and to identify evidence of artificial selection. The results indicated classification of a small number of predominant haplogroups for each gene and important insights into possible allelic diversity for each gene within the context of known causative mutations. The software has both a stand-alone and web-based version and can be used to analyze other genes, examine additional soybean datasets, and view similar genome sequence and SNP datasets from other species. PMID:24727730

  1. Microarray gene expression analysis reveals major differences between Toxocara canis and Toxocara cati neurotoxocarosis and involvement of T. canis in lipid biosynthetic processes.

    PubMed

    Janecek, Elisabeth; Wilk, Esther; Schughart, Klaus; Geffers, Robert; Strube, Christina

    2015-06-01

    Toxocara canis and Toxocara cati are globally occurring intestinal nematodes of dogs and cats with a high zoonotic potential. Migrating larvae in the CNS of paratenic hosts, including humans, may cause neurotoxocarosis resulting in a variety of neurological symptoms. Toxocara canis exhibits a stronger affinity to the CNS than T. cati, causing more severe neurological symptoms in the mouse model. Pathomechanisms of neurotoxocarosis as well as host responses towards the respective parasite are mostly unknown. Therefore, the aim of this study was to characterise the pathogenesis at a transcriptional level using whole genome microarray expression analysis and identify differences and similarities between T. canis- and T. cati-infected brains. Microarray analysis was conducted in cerebra and cerebella of infected C57Bl/6J mice 42daysp.i. revealing more differentially transcribed genes for T. canis- than T. cati-infected brains. In cerebra and cerebella of T. canis-infected mice, a total of 2304 and 1954 differentially transcribed genes, respectively, were identified whereas 113 and 760 differentially transcribed genes were determined in cerebra and cerebella of T. cati-infected mice. Functional annotation analysis revealed major differences in host responses in terms of significantly enriched biological modules. Up-regulated genes were mainly associated with the terms "immune and defence response", "sensory perception" as well as "behaviour/taxis" retrieved from the Gene Ontology database. These observations indicate a strong immune response in both infection groups with T. cati-infected brains revealing less severe reactions. Down-regulated genes in T. canis-infected cerebra and cerebella revealed a significant enrichment for the Gene Ontology term "lipid/cholesterol biosynthetic process". Cholesterol is a highly abundant and important component in the brain, representing several functions. Disturbances of synthesis as well as concentration changes may lead to

  2. Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (Leishmania) major infection.

    PubMed

    Coelho, Eduardo Antonio Ferraz; Tavares, Carlos Alberto Pereira; Lima, Karla de Melo; Silva, Célio Lopes; Rodrigues, José Maciel; Fernandes, Ana Paula

    2006-05-01

    Heat shock proteins (HSPs) are highly conserved among different organisms. A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic and immunotherapeutic effects against Mycobacterium tuberculosis infection in mice. Here, BALB/c mice were immunized with mycobacterial DNA-hsp65 or with DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable microspheres (MHSP/TDM), and challenged with Leishmania (Leishmania) major. MHSP/TDM conferred protection against L. major infection, as indicated by a significant reduction of edema and parasite loads in infected tissues. Although high levels of interferon-gamma and low levels of interleukin (IL)-4 and IL-10 were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals immunized with MHSP/TDM displayed a consistent Th1 immune response, i.e., significantly higher levels of anti-soluble Leishmania antigen (SLA) immunoglobulin G (IgG)2a and low anti-SLA IgG1 antibodies. These findings indicate that encapsulated MHSP/TDM is more immunogenic than naked hsp65 DNA, and has great potential to improve vaccine effectiveness against leishmaniasis and tuberculosis.

  3. Multiscale imaging characterization of dopamine transporter knockout mice reveals regional alterations in spine density of medium spiny neurons.

    PubMed

    Berlanga, M L; Price, D L; Phung, B S; Giuly, R; Terada, M; Yamada, N; Cyr, M; Caron, M G; Laakso, A; Martone, M E; Ellisman, M H

    2011-05-16

    The dopamine transporter knockout (DAT KO) mouse is a model of chronic hyperdopaminergia used to study a wide range of neuropsychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), drug abuse, depression, and Parkinson's disease (PD). Early studies characterizing this mouse model revealed a subtle, but significant, decrease in the anterior striatal volume of DAT KO mice accompanied by a decrease in neuronal cell body numbers (Cyr et al., 2005). The present studies were conducted to examine medium spiny neuron (MSN) morphology by extending these earlier reports to include multiscale imaging studies using correlated light microscopy (LM) and electron microscopy (EM) techniques. Specifically, we set out to determine if chronic hyperdopaminergia results in quantifiable or qualitative changes in DAT KO mouse MSNs relative to wild-type (WT) littermates. Using Neurolucida Explorer's morphometric analysis, we measured spine density, dendritic length and synapse number at ages that correspond with the previously reported changes in striatal volume and progressive cell loss. Light microscopic analysis using Neurolucida tracings of photoconverted striatal MSNs revealed a highly localized loss of dendritic spines on the proximal portion of the dendrite (30 μm from the soma) in the DAT KO group. Next, thick sections containing MSN dendritic segments located at a distance of 20-60 μm from the cell soma, a region of the dendrite where spine density is reported to be the highest, were analyzed using electron microscope tomography (EMT). Because of the resolution limits of LM, the EM analysis was an extra measure taken to assure that our analysis included nearly all spines. Spine density measurements collected from the EMT data revealed only a modest decrease in the DAT KO group (n=3 mice) compared to age-matched WT controls (n=3 mice), a trend that supports the LM findings. Finally, a synaptic quantification using unbiased stereology did not

  4. A global synthesis reveals biodiversity loss as a major driver of ecosystem change.

    PubMed

    Hooper, David U; Adair, E Carol; Cardinale, Bradley J; Byrnes, Jarrett E K; Hungate, Bruce A; Matulich, Kristin L; Gonzalez, Andrew; Duffy, J Emmett; Gamfeldt, Lars; O'Connor, Mary I

    2012-05-02

    Evidence is mounting that extinctions are altering key processes important to the productivity and sustainability of Earth's ecosystems. Further species loss will accelerate change in ecosystem processes, but it is unclear how these effects compare to the direct effects of other forms of environmental change that are both driving diversity loss and altering ecosystem function. Here we use a suite of meta-analyses of published data to show that the effects of species loss on productivity and decomposition--two processes important in all ecosystems--are of comparable magnitude to the effects of many other global environmental changes. In experiments, intermediate levels of species loss (21-40%) reduced plant production by 5-10%, comparable to previously documented effects of ultraviolet radiation and climate warming. Higher levels of extinction (41-60%) had effects rivalling those of ozone, acidification, elevated CO(2) and nutrient pollution. At intermediate levels, species loss generally had equal or greater effects on decomposition than did elevated CO(2) and nitrogen addition. The identity of species lost also had a large effect on changes in productivity and decomposition, generating a wide range of plausible outcomes for extinction. Despite the need for more studies on interactive effects of diversity loss and environmental changes, our analyses clearly show that the ecosystem consequences of local species loss are as quantitatively significant as the direct effects of several global change stressors that have mobilized major international concern and remediation efforts.

  5. Landwater variation in four major river basins of the Indochina peninsula as revealed by GRACE

    NASA Astrophysics Data System (ADS)

    Yamamoto, K.; Fukuda, Y.; Nakaegawa, T.; Nishijima, J.

    2007-04-01

    We estimated mass variations in four major river basins the Mekong, Irrawaddy, Salween and Chao Phraya river basins of the Indochina Peninsula using the newly released GRACE (Gravity Recovery and Climate Experiment) monthly gravity field solutions of UTCSR RL02 (University of Texas at Austin, Center for Space Research Release 02), JPL RL02 (Jet Propulsion Laboratory Release 02) and GFZ RL03 (GeoForschungsZentrum Potsdam Release 03). The estimated variations were compared with that calculated from a numerical model. The results show that there is a good agreement between the GRACE estimations and the model calculation for the Mekong and Irrawaddy basins, while the aggreement for the Salween and Chao Phraya basins is poor, mainly due to the spatial scale of the areas concerned. The comparison over the combined area of the four river basins shows fairly good agreement, although there are small quantitative discrepancies. The amplitudes of the annual signals of the GRACE solutions are 0.9- to 1.4-fold larger than that of the hydrological model, and the phases are delayed about 1 month compared with the model signal. The phase differences are probably due to improper treatments of the groundwater storage process in the hydrological model, suggesting that the GRACE data possibly provide constraints to the model parameters.

  6. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Facchini, Peter J

    2012-10-01

    Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3'-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3'-hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3'-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine.

  7. A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type 1.

    PubMed

    Madaule, P; Gairin, J E; Bénichou, S; Rossier, J

    1991-04-01

    In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10(4) clones) was first constructed in a new expression/secretion vector, using as inserts small fragments of HIV-1 DNA (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomyces cerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti-HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screening the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available.

  8. Phenotyping pipeline reveals major seedling root growth QTL in hexaploid wheat

    PubMed Central

    Atkinson, Jonathan A.; Wingen, Luzie U.; Griffiths, Marcus; Pound, Michael P.; Gaju, Oorbessy; Foulkes, M. John; Le Gouis, Jacques; Griffiths, Simon; Bennett, Malcolm J.; King, Julie; Wells, Darren M.

    2015-01-01

    Seedling root traits of wheat (Triticum aestivum L.) have been shown to be important for efficient establishment and linked to mature plant traits such as height and yield. A root phenotyping pipeline, consisting of a germination paper-based screen combined with image segmentation and analysis software, was developed and used to characterize seedling traits in 94 doubled haploid progeny derived from a cross between the winter wheat cultivars Rialto and Savannah. Field experiments were conducted to measure mature plant height, grain yield, and nitrogen (N) uptake in three sites over 2 years. In total, 29 quantitative trait loci (QTLs) for seedling root traits were identified. Two QTLs for grain yield and N uptake co-localize with root QTLs on chromosomes 2B and 7D, respectively. Of the 29 root QTLs identified, 11 were found to co-localize on 6D, with four of these achieving highly significant logarithm of odds scores (>20). These results suggest the presence of a major-effect gene regulating seedling root vigour/growth on chromosome 6D. PMID:25740921

  9. Phenotyping pipeline reveals major seedling root growth QTL in hexaploid wheat.

    PubMed

    Atkinson, Jonathan A; Wingen, Luzie U; Griffiths, Marcus; Pound, Michael P; Gaju, Oorbessy; Foulkes, M John; Le Gouis, Jacques; Griffiths, Simon; Bennett, Malcolm J; King, Julie; Wells, Darren M

    2015-04-01

    Seedling root traits of wheat (Triticum aestivum L.) have been shown to be important for efficient establishment and linked to mature plant traits such as height and yield. A root phenotyping pipeline, consisting of a germination paper-based screen combined with image segmentation and analysis software, was developed and used to characterize seedling traits in 94 doubled haploid progeny derived from a cross between the winter wheat cultivars Rialto and Savannah. Field experiments were conducted to measure mature plant height, grain yield, and nitrogen (N) uptake in three sites over 2 years. In total, 29 quantitative trait loci (QTLs) for seedling root traits were identified. Two QTLs for grain yield and N uptake co-localize with root QTLs on chromosomes 2B and 7D, respectively. Of the 29 root QTLs identified, 11 were found to co-localize on 6D, with four of these achieving highly significant logarithm of odds scores (>20). These results suggest the presence of a major-effect gene regulating seedling root vigour/growth on chromosome 6D. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. Sympatric Asian felid phylogeography reveals a major Indochinese-Sundaic divergence.

    PubMed

    Luo, Shu-Jin; Zhang, Yue; Johnson, Warren E; Miao, Lin; Martelli, Paolo; Antunes, Agostinho; Smith, James L D; O'Brien, Stephen J

    2014-04-01

    The dynamic geological and climatological history of Southeast Asia has spawned a complex array of ecosystems and 12 of the 37 known cat species, making it the most felid-rich region in the world. To examine the evolutionary histories of these poorly studied fauna, we compared phylogeography of six species (leopard cat Prionailurus bengalensis, fishing cat P. viverrinus, Asiatic golden cat Pardofelis temminckii, marbled cat P. marmorata, tiger Panthera tigris and leopard P. pardus) by sequencing over 5 kb of DNA each from 445 specimens at multiple loci of mtDNA, Y and X chromosomes. All species except the leopard displayed significant phylogenetic partitions between Indochina and Sundaland, with the central Thai-Malay Peninsula serving as the biogeographic boundary. Concordant mtDNA and nuclear DNA genealogies revealed deep Indochinese-Sundaic divergences around 2 MYA in both P. bengalensis and P. marmorata comparable to previously described interspecific distances within Felidae. The divergence coincided with serial sea level rises during the late Pliocene and early Pleistocene, and was probably reinforced by repeated isolation events associated with environmental changes throughout the Pleistocene. Indochinese-Sundaic differentiations within P. tigris and P. temminckii were more recent at 72-108 and 250-1570 kya, respectively. Overall, these results illuminate unexpected, deep vicariance events in Southeast Asian felids and provide compelling evidence of species-level distinction between the Indochinese and Sundaic populations in the leopard cat and marbled cat. Broader sampling and further molecular and morphometric analyses of these species will be instrumental in defining conservation units and effectively preserving Southeast Asian biodiversity.

  11. Comparative genomics reveals distinct host-interacting traits of three major human-associated propionibacteria.

    PubMed

    Mak, Tim N; Schmid, Monika; Brzuszkiewicz, Elzbieta; Zeng, Guanghong; Meyer, Rikke; Sfanos, Karen S; Brinkmann, Volker; Meyer, Thomas F; Brüggemann, Holger

    2013-09-22

    Propionibacteria are part of the human microbiota. Many studies have addressed the predominant colonizer of sebaceous follicles of the skin, Propionibacterium acnes, and investigated its association with the skin disorder acne vulgaris, and lately with prostate cancer. Much less is known about two other propionibacterial species frequently found on human tissue sites, Propionibacterium granulosum and Propionibacterium avidum. Here we analyzed two and three genomes of P. granulosum and P. avidum, respectively, and compared them to two genomes of P. acnes; we further highlight differences among the three cutaneous species with proteomic and microscopy approaches. Electron and atomic force microscopy revealed an exopolysaccharide (EPS)-like structure surrounding P. avidum cells, that is absent in P. acnes and P. granulosum. In contrast, P. granulosum possesses pili-like appendices, which was confirmed by surface proteome analysis. The corresponding genes were identified; they are clustered with genes encoding sortases. Both, P. granulosum and P. avidum lack surface or secreted proteins for predicted host-interacting factors of P. acnes, including several CAMP factors, sialidases, dermatan-sulphate adhesins, hyaluronidase and a SH3 domain-containing lipoprotein; accordingly, only P. acnes exhibits neuraminidase and hyaluronidase activities. These functions are encoded on previously unrecognized island-like regions in the genome of P. acnes. Despite their omnipresence on human skin little is known about the role of cutaneous propionibacteria. All three species are associated with a variety of diseases, including postoperative and device-related abscesses and infections. We showed that the three organisms have evolved distinct features to interact with their human host. Whereas P. avidum and P. granulosum produce an EPS-like surface structure and pili-like appendices, respectively, P. acnes possesses a number of unique surface-exposed proteins with host

  12. Genomic analysis reveals the major driving forces of bacterial life in the rhizosphere

    PubMed Central

    Matilla, Miguel A; Espinosa-Urgel, Manuel; Rodríguez-Herva, José J; Ramos, Juan L; Ramos-González, María Isabel

    2007-01-01

    Background Mutualistic interactions less well known than those between rhizobia and legumes are commonly found between plants and bacteria, frequently pseudomonads, which colonize roots and adjacent soil areas (the rhizosphere). Results A global analysis of Pseudomonas putida genes expressed during their interaction with maize roots revealed how a bacterial population adjusts its genetic program to this lifestyle. Differentially expressed genes were identified by comparing rhizosphere-colonizing populations with three distinct controls covering a variety of nutrients, growth phases and life styles (planktonic and sessile). Ninety rhizosphere up-regulated (rup) genes, which were induced relative to all three controls, were identified, whereas there was no repressed gene in common between the experiments. Genes involved in amino acid uptake and metabolism of aromatic compounds were preferentially expressed in the rhizosphere, which reflects the availability of particular nutrients in root exudates. The induction of efflux pumps and enzymes for glutathione metabolism indicates that adaptation to adverse conditions and stress (oxidative) response are crucial for bacterial life in this environment. The finding of a GGDEF/EAL domain response regulator among the induced genes suggests a role for the turnover of the secondary messenger c-diGMP in root colonization. Several mutants in rup genes showed reduced fitness in competitive root colonization. Conclusion Our results show the importance of two selective forces of different nature to colonize the rhizosphere: stress adaptation and availability of particular nutrients. We also identify new traits conferring bacterial survival in this niche and open a way to the characterization of specific signalling and regulatory processes governing the plant-Pseudomonas association. PMID:17784941

  13. A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes.

    PubMed

    Palomäki, Jaana; Sund, Jukka; Vippola, Minnamari; Kinaret, Pia; Greco, Dario; Savolainen, Kai; Puustinen, Anne; Alenius, Harri

    2015-01-01

    Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

  14. Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.

    PubMed

    Seebohm, B; Matinmehr, F; Köhler, J; Francino, A; Navarro-Lopéz, F; Perrot, A; Ozcelik, C; McKenna, W J; Brenner, B; Kraft, T

    2009-08-05

    The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the beta-myosin heavy chain, 723Arg --> Gly (R723G) and 736Ile --> Thr (I736T), in comparison to 719Arg --> Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S( *)(rigor R723G) = 0.84 pN/nm S( *)(rigor WT) = 0.29 pN/nm); and ii), a significant increase in force per head (F( *)(10 degrees C), 1.99 pN vs. 1.49 pN = 1.3-fold increase; F( *)(20 degrees C), 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S( *)(active10 degrees C), 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S( *)(rigor); 1.8-fold increase in F( *)(10 degrees C), 1.6-fold in F( *)(20 degrees C); twofold increase in S( *)(active10 degrees C)). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous

  15. PTT analysis of polyps from FAP patients reveals a great majority of APC truncating mutations

    SciTech Connect

    Luijt, R.B. van der; Khan, P.M.; Tops, C.M.J.

    1994-09-01

    The adenomatous polyposis coli (APC) gene plays an important role in colorectal carcinogenesis. Germline APC mutations are associated with familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer, characterized by the development of numerous adenomatous polyps in the large intestine. In order to investigate whether somatic inactivation of the remaining APC allele is necessary for adenoma formation, we collected multiple adenomatous polyps from individual FAP patients and investigated the presence of somatic mutations in the APC gene. The analysis of somatic APC mutations in these tumor samples was performed using a rapid and sensitive assay, called the protein truncation test (PTT). Chain-terminating somatic APC mutations were detected in the great majority of the tumor samples investigated. As expected, these mutations were mainly located in the mutation cluster region (MCR) in exon 15. Our results confirm that somatic mutation of the second APC allele is required for adenoma formation in FAP. Interestingly, in the polyps investigated in our study, the second APC allele is somatically inactivated through point mutation leading to a stop codon rather than by loss of heterozygosity. The observation that somatic second hits in APC are required for tumor development in FAP is in apparent accordance with the Knudson hypothesis for classical tumor suppressor genes. However, it is yet unknown whether chain-terminating APC mutations lead to a truncated protein exerting a dominant-negative effect or whether these mutations result in a null allele. Further investigation of this important issue will hopefully provide a better understanding of the mechanism of action of the mutated APC alleles in colorectal carcinogenesis.

  16. Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora.

    PubMed

    Steige, Kim A; Laenen, Benjamin; Reimegård, Johan; Scofield, Douglas G; Slotte, Tanja

    2017-01-31

    Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species.

  17. Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora

    PubMed Central

    Steige, Kim A.; Laenen, Benjamin; Reimegård, Johan; Slotte, Tanja

    2017-01-01

    Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species. PMID:28096395

  18. Study of fluid dynamics reveals direct communications between lymphatic vessels and venous blood vessels at lymph nodes of mice.

    PubMed

    Takeda, Kazu; Mori, Shiro; Kodama, Tetsuya

    2017-06-01

    Cancer cells metastasize to lymph nodes, with distant metastasis resulting in poor prognosis. The role of lymph node metastasis (LNM) in the spread of cancer to distant organs remain incompletely characterized. The visualization of flow dynamics in the lymphatic and blood vessels of MXH10/Mo-lpr/lpr mice, which develop systemic swelling of lymph nodes up to 10mm in diameter, has revealed that lymph nodes have the potential to be a direct source of systemic metastasis. However, it is not known whether these fluid dynamics characteristics are universal phenomena present in other strains of laboratory mice. Here we show that the fluid dynamics observed in MXH10/Mo-lpr/lpr mice are the same as those observed in C57BL/6J, BALB/cAJcl and NOD/ShiJic-scidJcl mice. Furthermore, when fluorescent solution was injected into a tumor-bearing lymph node, the flow dynamics observed in the efferent lymphatic vessels and thoracoepigastric vein depended on the type of tumor cell. Our results indicate that fluid dynamics in the lymphatic and blood vessels of MXH10/Mo-lpr/lpr mice are generalized phenomena seen in conventional laboratory mice. We anticipate our results can facilitate studies of the progression of lymphatic metastasis to hematogenous metastasis via lymph nodes and the early diagnosis and treatment of LNM. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Species specificity and augmentation of responses to class II major histocompatibility complex molecules in human CD4 transgenic mice

    PubMed Central

    1992-01-01

    Murine T cell responses to human class II major histocompatibility complex (MHC) molecules were shown to be a minimum of 20-70-fold lower than responses to allogeneic molecules. Transgenic mice expressing slightly below normal (75-95%) or very high (250-380%) cell surface levels of human CD4 were utilized to determine whether this was due to a species-specific interaction between murine CD4 and class II molecules. Human CD4 was shown to function in signal transduction events in murine T cells based on the ability of anti-human CD4 antibody to synergize with suboptimal doses of anti-murine CD3 antibody in stimulating T cell proliferation. In mice expressing lower levels of human CD4, T cell responses to human class II molecules were enhanced up to threefold, whereas allogeneic responses were unaltered. In mice expressing high levels of human CD4, responses to human class II molecules were enhanced at least 10-fold, whereas allogeneic responses were between one and three times the level of normal responses. The relatively greater enhancement of the response to human class II molecules in both lines argues for a preferential interaction between human CD4 and human class II molecules. In mice expressing lower levels of human CD4, responses to human class II molecules were blocked by antibodies to CD4 of either species, indicating participation by both molecules. In mice expressing high levels of human CD4, responses to both human and murine class II molecules were almost completely blocked with anti-human CD4 antibody, whereas anti-murine CD4 antibody had no effect. However, anti-murine CD4 continued to synergize with anti-CD3 in stimulating T cell proliferation in these mice. Thus, overexpression of human CD4 selectively impaired the ability of murine CD4 to assist in the process of antigen recognition. The ability of human CD4 to support a strong allogeneic response under these conditions indicates that this molecule can interact with murine class II molecules to a

  20. Tap water isotopes reveal the San Francisco Bay Area's plumbing and responses to a major drought

    NASA Astrophysics Data System (ADS)

    Tipple, B. J.; Jameel, M. Y.; Chau, T. H.; Mancuso, C. J.; Bowen, G. J.; Dufour, A.; Chesson, L. A.; Ehleringer, J. R.

    2016-12-01

    Water availability and sustainability in the Western United States is a major flashpoint among expanding communities, growing industries, and productive agricultural lands. This issue came to a head in 2015 in the State of California, when the State mandated a 25% reduction in urban water use following a multi-year drought that significantly depleted water resources. The demands for and challenges in supplying water are only expected to intensify as climate perturbations, such as the 2012-2015 California Drought, become more common. As a consequence, there is an increased need to understand linkages between population centers, water transport and usage, and the impacts of climate change on water resources and infrastructure. To better understand these relationships within a megalopolis in the Western United States, we collected and analyzed 723 tap waters from the San Francisco Bay Area during seven collection campaigns across 21 months during 2013-2015. San Francisco Bay Area was selected as it has well-known water management strategies and its water resources were dramatically affected by the 2012-2105 drought. Consistent with known water management strategies and previous reports of tap water isotope values, we found large spatiotemporal variations in the δ2H and δ18O values of tap waters, indicative of complex water transport systems and municipality-scale management decisions. We observed δ2H and δ18O values of tap water consistent with waters originating from snowmelt from the Sierra Nevada Mountains, local precipitation, ground water, and partially evaporated reservoir sources. A cluster analysis of measured tap water data grouped waters from 43 static sampling sites that were associated with specific water utility providers within the San Francisco Bay Area and known management practices. Water management responses to the drought, such as source switching, bringing in new sources, and conservation, could be observed within the isotope data from each of

  1. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

    PubMed

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

    2016-02-18

    Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

  2. Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome

    PubMed Central

    Furio, Laetitia; de Veer, Simon; Jaillet, Madeleine; Briot, Anais; Robin, Aurelie; Deraison, Celine

    2014-01-01

    Netherton syndrome (NS) is a severe genetic skin disease in which absence of a key protease inhibitor causes congenital exfoliative erythroderma, eczematous-like lesions, and atopic manifestations. Several proteases are overactive in NS, including kallikrein-related peptidase (KLK) 5, KLK7, and elastase-2 (ELA2), which are suggested to be part of a proteolytic cascade initiated by KLK5. To address the role of KLK5 in NS, we have generated a new transgenic murine model expressing human KLK5 in the granular layer of the epidermis (Tg-KLK5). Transgene expression resulted in increased proteolytic activity attributable to KLK5 and its downstream targets KLK7, KLK14, and ELA2. Tg-KLK5 mice developed an exfoliative erythroderma with scaling, growth delay, and hair abnormalities. The skin barrier was defective and the stratum corneum was detached through desmosomal cleavage. Importantly, Tg-KLK5 mice displayed cutaneous and systemic hallmarks of severe inflammation and allergy with pruritus. The skin showed enhanced expression of inflammatory cytokines and chemokines, infiltration of immune cells, and markers of Th2/Th17/Th22 T cell responses. Moreover, serum IgE and Tslp levels were elevated. Our study identifies KLK5 as an important contributor to the NS proteolytic cascade and provides a new and viable model for the evaluation of future targeted therapies for NS or related diseases such as atopic dermatitis. PMID:24534191

  3. Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites

    PubMed Central

    Maroto, Beatriz; Ramírez, Juan C.; Almendral, José M.

    2000-01-01

    The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the 32P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily

  4. Depletion of peritoneal CD5+ B cells has no effect on the course of Leishmania major infection in susceptible and resistant mice

    PubMed Central

    BABAI, B; LOUZIR, H; CAZENAVE, P -A; DELLAGI, K

    1999-01-01

    The mouse peritoneal cavity contains a unique self-renewing population of B cells (B-1) derived from fetal liver precursors and mainly producing polyreactive antibodies. Since B-1 cells are a potential source of IL-10, it has been suggested that these cells may contribute to the susceptibility of BALB/c mice to Leishmania major infection by skewing the T helper cell network towards a Th2 phenotype. Accordingly, L. major infection of B cell-defective BALB/c Xid mice (lacking B-1 cells) induces less severe disease compared with controls. However, in addition to the lack of B-1 cells, the Xid immune deficiency is characterized by high endogenous interferon-gamma (IFN-γ) production. In the present study, the role of B-1 cells during L. major infection was investigated in mice experimentally depleted of peritoneal B-1 cells. Six weeks old C57Bl/6 and BALB/c mice were lethally irradiated and reconstituted with autologous bone marrow which allows systemic depletion of B-1 cells. Untreated BALB/c, C57Bl/6 as well as BALB/c Xid mice were used as controls. After reconstitution, mice were injected with L. major amastigotes and progression was followed using clinical, parasitological and immunological criteria. As previously reported, BALB/c Xid mice showed a significant reduction in disease progression. In contrast, despite the dramatic reduction of B-1 cells, B-1-depleted BALB/c mice showed similar or even worse disease progression compared with control BALB/c mice. No differences were found between B-1-depleted or control C57Bl/6 mice. Our data suggest that the B-1 cells do not contribute to the susceptibility of BALB/c mice to L. major infection. PMID:10403925

  5. The opioid system majorly contributes to preference for fat emulsions but not sucrose solutions in mice.

    PubMed

    Sakamoto, Kazuhiro; Okahashi, Tatsuya; Matsumura, Shigenobu; Okafuji, Yoko; Adachi, Shin-ichi; Tsuzuki, Satoshi; Inoue, Kazuo; Fushiki, Tohru

    2015-01-01

    Rodents show a stronger preference for fat than sucrose, even if their diet is isocaloric. This implies that the preference mechanisms for fat and sucrose differ. To compare the contribution of the opioid system to the preference of fat and sucrose, we examined the effects of mu-, delta-, kappa-, and non-selective opioid receptor antagonists on the preference of sucrose and fat, assessed by a two-bottle choice test and a licking test, in mice naïve to sucrose and fat ingestion. Administration of non-selective and mu-selective opioid receptor antagonists more strongly inhibited the preference of fat than sucrose. While the preference of fat was reduced to the same level as water by the antagonist administration that of sucrose was still greater than water. Our results suggest that the preference of fat relies strongly on the opioid system, while that of sucrose is regulated by other mechanisms in addition to the opioid system.

  6. Mapping of Craniofacial Traits in Outbred Mice Identifies Major Developmental Genes Involved in Shape Determination

    PubMed Central

    Pallares, Luisa F.; Carbonetto, Peter; Gopalakrishnan, Shyam; Parker, Clarissa C.; Ackert-Bicknell, Cheryl L.; Palmer, Abraham A.; Tautz, Diethard

    2015-01-01

    The vertebrate cranium is a prime example of the high evolvability of complex traits. While evidence of genes and developmental pathways underlying craniofacial shape determination is accumulating, we are still far from understanding how such variation at the genetic level is translated into craniofacial shape variation. Here we used 3D geometric morphometrics to map genes involved in shape determination in a population of outbred mice (Carworth Farms White, or CFW). We defined shape traits via principal component analysis of 3D skull and mandible measurements. We mapped genetic loci associated with shape traits at ~80,000 candidate single nucleotide polymorphisms in ~700 male mice. We found that craniofacial shape and size are highly heritable, polygenic traits. Despite the polygenic nature of the traits, we identified 17 loci that explain variation in skull shape, and 8 loci associated with variation in mandible shape. Together, the associated variants account for 11.4% of skull and 4.4% of mandible shape variation, however, the total additive genetic variance associated with phenotypic variation was estimated in ~45%. Candidate genes within the associated loci have known roles in craniofacial development; this includes 6 transcription factors and several regulators of bone developmental pathways. One gene, Mn1, has an unusually large effect on shape variation in our study. A knockout of this gene was previously shown to affect negatively the development of membranous bones of the cranial skeleton, and evolutionary analysis shows that the gene has arisen at the base of the bony vertebrates (Eutelostomi), where the ossified head first appeared. Therefore, Mn1 emerges as a key gene for both skull formation and within-population shape variation. Our study shows that it is possible to identify important developmental genes through genome-wide mapping of high-dimensional shape features in an outbred population. PMID:26523602

  7. Cognitive decline following major surgery is associated with gliosis, β-amyloid accumulation, and τ phosphorylation in old mice.

    PubMed

    Wan, Yanjie; Xu, Jing; Meng, Fanzhen; Bao, Yuhua; Ge, Yeying; Lobo, Niyati; Vizcaychipi, Marcela P; Zhang, Denghai; Gentleman, Steve M; Maze, Mervyn; Ma, Daqing

    2010-11-01

    Elderly patients undergoing major surgery often develop cognitive dysfunction and the mechanism of this postoperative complication remains elusive. We sought to determine whether postoperative cognitive dysfunction in old mice is associated with the pathogenesis of Alzheimer's disease. Prospective, randomized study. University teaching hospital-based research laboratory. One-hundred and twenty C57BL/6 14-mo-old male mice (weighing 30-40 g). Mice received intraperitoneal injections of either vehicle or Celastrol (a potent anti-inflammatory compound) for 3 days before undergoing sham surgery or partial hepatectomy, on the surgery day, and for a further 4 days after surgery. Cognitive function, hippocampal neuroinflammation, and pathologic markers of Alzheimer's disease were assessed 1 day after surgery day 1, 3, or 7. Cognitive impairment following surgery was associated with the appearance of certain pathologic hallmarks of Alzheimer's disease: microgliosis, astrogliosis, enhanced transcriptional and translational activity of β-amyloid precursor protein, β-amyloid production, and τ protein hyperphosphorylation in the hippocampus. Surgery-induced changes in cognitive dysfunction were prevented by the administration of Celastrol as were changes in β-amyloid and τ processing. These data suggest that surgery can provoke astrogliosis, β-amyloid accumulation, and τ phosphorylation in old subjects, which is likely to be associated with the cognitive decline seen in postoperative cognitive dysfunction.

  8. Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

    PubMed Central

    Tourne, S; van Santen, H M; van Roon, M; Berns, A; Benoist, C; Mathis, D; Ploegh, H

    1996-01-01

    Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice. Images Fig. 1 Fig. 2 PMID:8643655

  9. Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response

    PubMed Central

    1995-01-01

    Mice with homologous disruption of the gene coding for the ligand- binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN- gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells. PMID:7869054

  10. Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors.

    PubMed

    Heck, Detlef H; Zhao, Yu; Roy, Snigdha; LeDoux, Mark S; Reiter, Lawrence T

    2008-07-15

    Angelman syndrome (AS) is a childhood-onset neurogenetic disorder characterized by functionally severe developmental delay with mental retardation, deficits in expressive language, ataxia, appendicular action tremors and unique behaviors such as inappropriate laughter and stimulus-sensitive hyperexcitibility. Most cases of AS are caused by mutations which disrupt expression of maternal UBE3A. Although some progress has been made in understanding hippocampal-related memory and learning aspects of the disorder using Ube3a deficient mice, the numerous motoric abnormalities associated with AS (ataxia, action tremor, dysarthria, dysphagia, sialorrhea and excessive chewing/mouthing behaviors) have not been fully explored with mouse models. Here we use a novel quantifiable analysis of fluid consumption and licking behavior along with a battery of motor tests to examine cerebellar and other motor system defects in Ube3a deficient mice. Mice with a maternally inherited Ube3a deficiency (Ube3a(m-/p+)) show defects in fluid consumption behavior which are different from Ube3a(m-/p-) mice. The rhythm of fluid licking and number of licks per visit were significantly different among the three groups (m-/p-, m-/p+, m+/p+) and indicate that not only was fluid consumption dependent on Ube3a expression in the cerebellum, but may also depend on low levels of Ube3a expression in other brain regions. Additional neurological testing revealed defects in both Ube3a(m-/p+) and Ube3a(m-/p-) mice in rope climbing, grip strength, gait and a raised-beam task. Long-term observation of fluid consumption behavior is the first phenotype reported that differentiates between mice with a maternal loss of function versus complete loss of Ube3a in the brain. The neuronal and molecular mechanisms underlying mouse fluid consumption defects specifically associated with maternally inherited Ube3a deficiency may reveal important new insights into the pathobiology of AS in humans.

  11. Differential Binding between Volatile Ligands and Major Urinary Proteins Due to Genetic Variation in Mice

    DTIC Science & Technology

    2012-06-20

    Kruskal– Wallis One-way ANOVA). As shown in Table 1, the degree of release in the majority of the ligands is different between strains, indicating that...0.3309 Cedrol 28.5 150 2.65 13.18b 5.57b 0.0003 a Comparing fold differences across inbred strains (Kruskal– Wallis One-way ANOVA). b Significant...volatile ligands associ- ating with the major urinary protein. Proc Biol Sci 1999;266: 2017 –22. [10] Hurst JL, Robertson DHL, Tolladay U, Beynon RJ

  12. Pam heterozygous mice reveal essential role for Cu in amygdalar behavioral and synaptic function

    PubMed Central

    Gaier, Eric D; Eipper, Betty A; Mains, Richard E

    2014-01-01

    Cu is an essential element with many biological roles, but its roles in the mammalian nervous system are poorly understood. Mice deficient in the cuproenzyme peptidylglycine α-amidating monooxygenase (PAM+/− mice) were initially generated to study neuropeptide amidation. PAM+/− mice exhibited profound deficits in a few behavioral tasks, including enhancements in innate fear along with deficits in acquired fear. Interestingly, several PAM+/− phenotypes were recapitulated in Cu restricted wildtype mice and rescued in Cu supplemented PAM+/− mice. These behaviors correspond to enhanced excitability and deficient synaptic plasticity in the amygdala of PAM+/− mice, which are also rescued by Cu supplementation. Cu and ATP7A are present at synapses, in key positions to respond to and influence synaptic activity. Further study demonstrated that extracellular Cu is necessary for wildtype synaptic plasticity and sufficient to rescue PAM+/− LTP. These experiments support roles for PAM in Cu homeostasis and for synaptic Cu in amygdalar function. PMID:24593825

  13. Transcriptome profiling reveals divergent expression shifts in brown and white adipose tissue from long-lived GHRKO mice

    PubMed Central

    Zhi, Xu; Rohde, Kyle; List, Edward O.; Berryman, Darlene E.; Kopchick, John J.; Gesing, Adam; Fang, Yimin; Masternak, Michal M.

    2015-01-01

    Mice lacking the growth hormone receptor (GHRKO) exhibit improved lifespan and healthspan due to loss of growth hormone signaling. Both the distribution and activity of brown and white adipose tissue (BAT and WAT) are altered in GHRKO mice, but the contribution of each tissue to age-related phenotypes has remained unclear. We therefore used whole-genome microarrays to evaluate transcriptional differences in BAT and WAT depots between GHRKO and normal littermates at six months of age. Our findings reveal a unique BAT transcriptome as well as distinctive responses of BAT to Ghr ablation. BAT from GHRKO mice exhibited elevated expression of genes associated with mitochondria and metabolism, along with reduced expression of genes expressed by monocyte-derived cells (dendritic cells [DC] and macrophages). Largely the opposite was observed in WAT, with increased expression of DC-expressed genes and reduced expression of genes associated with metabolism, cellular respiration and the mitochondrial inner envelope. These findings demonstrate divergent response patterns of BAT and WAT to loss of GH signaling in GHRKO mice. These patterns suggest both BAT and WAT contribute in different ways to phenotypes in GHRKO mice, with Ghr ablation blunting inflammation in BAT as well as cellular metabolism and mitochondrial biogenesis in WAT. PMID:26436954

  14. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    PubMed

    Sheehan, Michael J; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J; Hurst, Jane L; Nachman, Michael W

    2016-03-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  15. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice

    PubMed Central

    Sheehan, Michael J.; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J.; Hurst, Jane L.; Nachman, Michael W.

    2016-01-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones. PMID:26938775

  16. Evaluation of Apoptotic and Antileishmanial Activities of Artemisinin on Promastigotes and BALB/C Mice Infected with Leishmania major

    PubMed Central

    GHAFFARIFAR, Fatemeh; ESAVAND HEYDARI, Farzad; DALIMI, Abdolhosein; HASSAN, Zuhair M.; DELAVARI, Mahdi; MIKAEILOO, Hajar

    2015-01-01

    Background: In leishmaniasis, some drugs prescribed for treatment have toxic effects and there are reports about drug resistance in some countries. Due to this fact, using herbal drugs such as artemisinin with good efficacy and low toxic effect might be suitable. Methods: We evaluated the apoptotic effect of artemisinin on Leishmania major in vitro and the antileishmanial activities of artemisinin on leishmaniasis in BALB/c mice and at the end INF-γ and IL-4 cytokines levels were detected by ELISA in spleen cell culture supernatants. During treatment the lesion size and survival rate were measured each four and ten days, respectively. Results: Percentage of early and late apoptosis in promastigotes of control group and promastigotes treated with 10, 25, 50 and 100 μg/ml of artemisinin after 48 h were 0.13, 16.04, 41.23, 49.03 and 81.83, respectively. The IFN-γ in ointment treated group were higher than those of other groups (P<0.05). The in vivo results showed that ointment compounds healed the lesions more effectively rather than intraperitoneal injection method (P<0.05). The survival rate of mice 150 days after challenge in treated group with ointment of artemisinin was 66% while all mice in control groups were died. Conclusion: All of in vitro results represented that this drug had antileishmanial effects and these results were confirmed by evaluation effects in vivo condition of leishmaniasis. Interestingly, according to these results it can be concluded that this drug has antileishmanial effects in vitro and in vivo conditions. Artemisinin induces cytotoxic effect on L. major via apoptosis-related mechanism. PMID:26246824

  17. Effects of combined therapy with silymarin and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice.

    PubMed

    Jabini, R; Jaafari, M R; Vahdati Hasani, F; Ghazizadeh, F; Khamesipour, A; Karimi, G

    2015-03-01

    Leishmania major is resistant to the traditional treatments in many parts of the world. PgpA, a member of (ABC) transporter superfamily, has been identified in Leishmania involved in antimony resistance. Silymarin can inhibit PgpA. The aim of this study was to determine the effect of combined therapy with glucantime and silymarin on Cutaneous Leishmaniasis. The effects of silymarin on response of L. major to glucantime were evaluated with amastigote macrophage and mice model of leishmaniasis. Immediately after injection in mice inoculated into footpads with L. major amastigote, systemic treatment was performed and the size of footpad swelling was measured twice a week. 4 and 8 weeks after the beginning of the treatment, splenic parasite burden was done. Silymarin showed no significant effect on the response of L. major promastigotes to glucantime. 2 formulations (glucantime 25 µm with silymarin 25 µm or 12.5 µm) reduced cell death in amastigote assays. The effect of silymarin on footpad swelling was detected when the combination of low-dose glucantime (20 mg/kg) with 25-50 mg/kg silymarin (especially 50 mg/kg) were used at day 22 of post infection (P<0.05). According to the parasite burden data, use of silymarin in the presence of different doses of glucantime, did not show significant effect compared to glucantime alone. The results of this study suggest that silymarin in conjunction with glucantime may have benefit effects in murine model of cutaneous leishmaniasis. © Georg Thieme Verlag KG Stuttgart · New York.

  18. The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-02-01

    Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

  19. An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates

    PubMed Central

    Sun, Ping; Clamp, John; Xu, Dapeng; Huang, Bangqin; Shin, Mann Kyoon

    2016-01-01

    Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes. PMID:26880590

  20. An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates.

    PubMed

    Sun, Ping; Clamp, John; Xu, Dapeng; Huang, Bangqin; Shin, Mann Kyoon

    2016-02-16

    Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes.

  1. Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion.

    PubMed

    Di Cosmo, Caterina; Liao, Xiao-Hui; Dumitrescu, Alexandra M; Philp, Nancy J; Weiss, Roy E; Refetoff, Samuel

    2010-09-01

    The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid-precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients.

  2. Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion

    PubMed Central

    Di Cosmo, Caterina; Liao, Xiao-Hui; Dumitrescu, Alexandra M.; Philp, Nancy J.; Weiss, Roy E.; Refetoff, Samuel

    2010-01-01

    The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid–precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients. PMID:20679730

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

    PubMed

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

    2015-11-12

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

  4. Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

    PubMed Central

    Tremblay, Jacqueline M.; Oliveira, Sergio C.; Da’dara, Akram A.; Skelly, Patrick J.

    2017-01-01

    Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development. PMID:28095417

  5. Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits

    PubMed Central

    Heier, Christopher R.; Guerron, Alfredo D.; Korotcov, Alexandru; Lin, Stephen; Gordish-Dressman, Heather; Fricke, Stanley; Sze, Raymond W.; Hoffman, Eric P.; Wang, Paul; Nagaraju, Kanneboyina

    2014-01-01

    In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy. PMID:25390038

  6. Network hyperexcitability in hippocampal slices from Mecp2 mutant mice revealed by voltage-sensitive dye imaging

    PubMed Central

    Calfa, Gaston; Hablitz, John J.

    2011-01-01

    Dysfunctions of neuronal and network excitability have emerged as common features in disorders associated with intellectual disabilities, autism, and seizure activity, all common clinical manifestations of Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Here, we evaluated the consequences of Mecp2 mutation on hippocampal network excitability, as well as synapse structure and function using a combination of imaging and electrophysiological approaches in acute slices. Imaging the amplitude and spatiotemporal spread of neuronal depolarizations with voltage-sensitive dyes (VSD) revealed that the CA1 and CA3 regions of hippocampal slices from symptomatic male Mecp2 mutant mice are highly hyperexcitable. However, only the density of docked synaptic vesicles and the rate of release from the readily releasable pool are impaired in Mecp2 mutant mice, while synapse density and morphology are unaffected. The differences in network excitability were not observed in surgically isolated CA1 minislices, and blockade of GABAergic inhibition enhanced VSD signals to the same extent in Mecp2 mutant and wild-type mice, suggesting that network excitability originates in area CA3. Indeed, extracellular multiunit recordings revealed a higher level of spontaneous firing of CA3 pyramidal neurons in slices from symptomatic Mecp2 mutant mice. The neuromodulator adenosine reduced the amplitude and spatiotemporal spread of VSD signals evoked in CA1 of Mecp2 mutant slices to wild-type levels, suggesting its potential use as an anticonvulsant in RTT individuals. The present results suggest that hyperactive CA3 pyramidal neurons contribute to hippocampal dysfunction and possibly to limbic seizures observed in Mecp2 mutant mice and RTT individuals. PMID:21307327

  7. Novel Role for SGK3 in Glucose Homeostasis Revealed in SGK3/Akt2 Double-Null Mice

    PubMed Central

    Yao, Li-Jun; McCormick, James A.; Wang, Jian; Yang, Katherine Y.; Kidwai, Atif; Colussi, Gian Luca; Boini, Krishna M.; Birnbaum, Morris J.; Lang, Florian; German, Michael S.

    2011-01-01

    The phosphatidylinositol-3-kinase-dependent kinase, Akt2, plays a central role in mediating insulin effects in glucose-metabolizing tissues. Akt2 knockout mice display insulin resistance with a reactive increase in pancreatic islet mass and hyperinsulinemia. The related phosphatidylinositol-3-kinase-dependent kinase, serum- and glucocorticoid-regulated kinase 3 (SGK3), is essential for normal postnatal hair follicle development but plays no apparent role in glucose homeostasis. We report here an unexpected role of SGK3 in islet β-cell function, which is revealed in Akt2/SGK3 double-knockout (DKO) mice. DKO mice have markedly worse glucose homeostasis than Akt2 single-null animals, including greater baseline glucose, and greater rise in blood glucose after glucose challenge. However, surprisingly, our data strongly support the idea that this exacerbation of the glucose-handling defect is due to impaired β-cell function, rather than increased insulin resistance in peripheral tissues. DKO mice had lower plasma insulin and C-peptide levels, lower β-cell mass, reduced glucose-stimulated insulin secretion, and greater sensitivity to exogenous insulin than Akt2 single nulls. We further demonstrated that SGK3 is strongly expressed in normal mouse islets and, interestingly, that β-catenin expression is dramatically lower in the islets of DKO mice than in those of Akt2−/−/SGK3+/+ or Akt2−/−/SGK3+/− mice. Taken together, these data strongly suggest that SGK3 plays a previously unappreciated role in glucose homeostasis, likely through direct effects within β-cells, to stimulate proliferation and insulin release, at least in part by controlling the expression and activity of β-catenin. PMID:21980074

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  10. Use of topical liposomes containing meglumine antimoniate (Glucantime) for the treatment of L. major lesion in BALB/c mice.

    PubMed

    Kalat, S A Moosavian; Khamesipour, A; Bavarsad, N; Fallah, M; Khashayarmanesh, Z; Feizi, E; Neghabi, K; Abbasi, A; Jaafari, M R

    2014-08-01

    Treatment of cutaneous leishmaniasis (CL) is a dream for the patients, health care authorities and scientists. The aim of this study was to develop a topical liposomal meglumine antimoniate (MA, Glucantime™) (Lip-MA) formulation and evaluate the therapeutic effects of the preparation on lesion induced by Leishmania major in BALB/c mice. Liposomes containing 22.5% MA (6.4% Sb(+5)) with and without oleic acid (LMA-OA and LMA) were formulated using fusion method plus homogenization and characterized for the size and encapsulation efficiency. The penetration of MA from the LMA-OA and LMA formulations through and into the skin was checked in vitro using Franz diffusion cells fitted with mouse skin at 37°C for 8h. The in vitro permeation data showed that almost 1.5% of formulations applied in the mouse skin were penetrated and the amount retained in the skin was about 65%. The 50% effective dose of LMA and LMA-OA against amastigotes of L. major was 46.36 and 41.01 μg/ml, respectively. LMA or LMA-OA was used topically twice a day for 4 weeks to treat the lesion induced by L. major in susceptible BALB/c mice. The results showed a significantly (P<0.001) smaller lesion size in the treated groups of mice compared to the control groups which received either empty liposomes or phosphate-buffered saline (PBS). The spleen parasite burden was significantly (P<0.001) lower in the treated groups compared to the control groups receiving either empty liposomes or PBS at the end of the treatment period. However, when the treatment was stopped, the lesion size progressed and spleen parasite burden increased in LMA and LMA-OA groups, but still was significantly less than the control groups (P<0.05). There was no significant difference between the two formulations of LMA and LMA-OA. The results suggested that topical liposomes containing MA might be an appropriate choice for clinical trials for the treatment of CL. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice.

    PubMed

    Baudat, F; Buard, J; Grey, C; Fledel-Alon, A; Ober, C; Przeworski, M; Coop, G; de Massy, B

    2010-02-12

    Meiotic recombination events cluster into narrow segments of the genome, defined as hotspots. Here, we demonstrate that a major player for hotspot specification is the Prdm9 gene. First, two mouse strains that differ in hotspot usage are polymorphic for the zinc finger DNA binding array of PRDM9. Second, the human consensus PRDM9 allele is predicted to recognize the 13-mer motif enriched at human hotspots; this DNA binding specificity is verified by in vitro studies. Third, allelic variants of PRDM9 zinc fingers are significantly associated with variability in genome-wide hotspot usage among humans. Our results provide a molecular basis for the distribution of meiotic recombination in mammals, in which the binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.

  12. Plasmid-Encoded Pgp3 Is a Major Virulence Factor for Chlamydia muridarum To Induce Hydrosalpinx in Mice

    PubMed Central

    Liu, Yuanjun; Huang, Yumeng; Yang, Zhangsheng; Sun, Yina; Gong, Siqi; Hou, Shuping; Chen, Chaoqun; Li, Zhongyu; Liu, Quanzhong; Wu, Yimou; Baseman, Joel

    2014-01-01

    Hydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice. PMID:25287930

  13. Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains.

    PubMed

    Takeo, Toru; Nakagata, Naomi

    2016-09-15

    Improvement of the superovulation technique will help to enhance the efficiency of embryo and animal production. Blocking inhibin using inhibin antiserum (IAS) is known to promote follicular development by increasing the level of FSH. Previously, we reported that coadministration of IAS and eCG produced more than 100 oocytes from a single female C57BL/6 mouse at 4 weeks old. The oocytes derived from the IAS + eCG (IASe) treatment were able to fertilize and develop normally into offspring. In this study, we examined the effect of IASe treatment on the numbers of ovulated oocytes in major inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J, and FVB/NJ) and outbred (CD1) mice strains at 4 weeks old. We confirmed the fertilization and developmental ability of the IASe-derived oocytes. IASe treatment ovulated 1.5 to 3.2 times higher numbers of oocytes than eCG treatment alone. The fertilization rate of IASe-derived oocytes was similar to that of eCG-derived oocytes. In vitro and in vivo developmental rates of the embryos derived from IASe were similar to the rates of embryos derived from eCG. We have shown that superovulation by IASe is very effective in obtaining high numbers of ovulated oocytes from small numbers of oocyte donor in a number of mice strains. The superovulation technique will contribute to the archiving of cryopreserved embryos of genetically engineered mice using small numbers of donors and has the potential to produce more live animals for rederivation of the archived mouse lines in mouse repositories. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. An adenoviral vector expressing lipoprotein A, a major antigen of Mycoplasma mycoides subspecies mycoides, elicits robust immune responses in mice.

    PubMed

    Carozza, Marlène; Rodrigues, Valérie; Unterfinger, Yves; Galea, Sandra; Coulpier, Muriel; Klonjkowski, Bernard; Thiaucourt, François; Totté, Philippe; Richardson, Jennifer

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP.

  15. The effect of low and high plasma levels of insulin-like growth factor-1 (IGF-1) on the morphology of major organs: studies of Laron dwarf and bovine growth hormone transgenic (bGHTg) mice.

    PubMed

    Piotrowska, Katarzyna; Borkowska, Sylwia J; Wiszniewska, Barbara; Laszczyńska, Maria; Słuczanowska-Głabowska, Sylwia; Havens, Aaron M; Kopchick, John J; Bartke, Andrzej; Taichman, Russel S; Kucia, Magda; Ratajczak, Mariusz Z

    2013-10-01

    It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals. To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on the morphology of major organs, we analyzed lung, heart, liver, kidney, bone marrow, and spleen isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice (with low circulating plasma levels of IGF-1) and 6-month-old bovine growth hormone transgenic (bGHTg) mice (with high circulating plasma levels of IGF-1). The ages of the two mutant strains employed in our studies were selected based on their overall ~50% survival (Laron dwarf mice live up to ~4 years and bGHTg mice up to ~1 year). Morphological analysis of the organs of long-living 2-year-old Laron dwarf mice revealed a lower biological age for their organs compared with normal littermates, with more brown adipose tissue (BAT) surrounding the main body organs, lower levels of steatosis in liver, and a lower incidence of leukocyte infiltration in different organs. By contrast, the organs of 6-month-old, short-living bGHTg mice displayed several abnormalities in liver and kidney and a reduced content of BAT around vital organs.

  16. Brain Transcriptional Responses to High-Fat Diet in Acads-Deficient Mice Reveal Energy Sensing Pathways

    PubMed Central

    Kruger, Claudia; Kumar, K. Ganesh; Mynatt, Randall L.; Volaufova, Julia; Richards, Brenda K.

    2012-01-01

    Background How signals from fatty acid metabolism are translated into changes in food intake remains unclear. Previously we reported that mice with a genetic inactivation of Acads (acyl-coenzyme A dehydrogenase, short-chain), the enzyme responsible for mitochondrial beta-oxidation of C4–C6 short-chain fatty acids (SCFAs), shift consumption away from fat and toward carbohydrate when offered a choice between diets. In the current study, we sought to indentify candidate genes and pathways underlying the effects of SCFA oxidation deficiency on food intake in Acads−/− mice. Methodology/Principal Findings We performed a transcriptional analysis of gene expression in brain tissue of Acads−/− and Acads+/+ mice fed either a high-fat (HF) or low-fat (LF) diet for 2 d. Ingenuity Pathway Analysis revealed three top-scoring pathways significantly modified by genotype or diet: oxidative phosphorylation, mitochondrial dysfunction, and CREB signaling in neurons. A comparison of statistically significant responses in HF Acads−/− vs. HF Acads+/+ (3917) and Acads+/+ HF vs. LF Acads+/+ (3879) revealed 2551 genes or approximately 65% in common between the two experimental comparisons. All but one of these genes were expressed in opposite direction with similar magnitude, demonstrating that HF-fed Acads-deficient mice display transcriptional responses that strongly resemble those of Acads+/+ mice fed LF diet. Intriguingly, genes involved in both AMP-kinase regulation and the neural control of food intake followed this pattern. Quantitative RT-PCR in hypothalamus confirmed the dysregulation of genes in these pathways. Western blotting showed an increase in hypothalamic AMP-kinase in Acads−/− mice and HF diet increased, a key protein in an energy-sensing cascade that responds to depletion of ATP. Conclusions Our results suggest that the decreased beta-oxidation of short-chain fatty acids in Acads-deficient mice fed HF diet produces a state of energy deficiency in the

  17. Metabonomic phenotyping reveals an embryotoxicity of deca-brominated diphenyl ether in mice.

    PubMed

    Chi, Yi; Xia, Hongfei; Su, Mingming; Song, Peipei; Qi, Xin; Cui, Yi; Cao, Yu; Chen, Tianlu; Qiu, Yunping; Zhao, Aihua; Ma, Xu; Zheng, Xiaoying; Jia, Wei

    2011-11-21

    Recent studies have demonstrated that polybrominated diphenyl ethers (PBDEs), a group of industrial chemicals, could disrupt thyroid hormone homeostasis and exhibit neurotoxicity, reproductive toxicity, and embryotoxicity. However, clear evidence of embryotoxicity and neurotoxicity of many of these congeners, such as deca-BDE, one of the least bioactive congeners of PBDEs, is still lacking. In the present study, we investigated deca-BDE embryotoxicity by quantitative analysis of two essential thyroid hormones (T4 and T3) and a variety of small-molecule metabolites in the serum of deca-BDE-dosed pregnant mice. Four groups of pregnant C57 mice were administrated with deca-BDE in 20% fat emulsion at a dose of 150, 750, 1,500, or 2,500 mg/kg body weight via gastric intubation on gestation days (g.d.s) 7 to 9, while a control group was given 20% fat emulsion. Maternal mice were euthanized on g.d. 16 and examined for external malformations of the fetus. Maternal serum samples were collected and analyzed by the enzyme linked immunosorbent assay (ELISA) and gas chromatography-time-of-flight mass spectrometry (GC-TOF MS). Using multivariate statistical analysis, we observed a significantly altered metabolic profile associated with deca-BDE embryotoxicity in maternal serum. Our results also demonstrated that deca-BDE at a dose of 2 500 mg/kg body weight induced significant disruption of thyroid hormone metabolism, the TCA cycle, and lipid metabolism in maternal mice, which subsequently led to a significant inhibition of fetal growth and development. We concluded that deca-BDE-induced embryotoxicity closely correlated with global metabolic disruption that can be characterized by thyroid hormone deficiency, disrupted lipid metabolism, and a depleted level of cholesterol in maternal mice.

  18. Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice.

    PubMed

    van der Merwe, Elizabeth L; Kidson, Susan H

    2016-05-01

    Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma.

  19. Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice

    PubMed Central

    Winkel, Angelika F.; Polack, James; Tang, Hui; Brachs, Maria; Margerie, Daniel; Brunner, Bodo; Jahn-Hofmann, Kerstin; Ruetten, Hartmut; Spranger, Joachim; Schmoll, Dieter

    2016-01-01

    The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti-oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice. PMID:27814396

  20. Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn.

    PubMed

    Gutierrez-Mecinas, Maria; Kuehn, Emily D; Abraira, Victoria E; Polgár, Erika; Watanabe, Masahiko; Todd, Andrew J

    2016-08-04

    The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

    PubMed Central

    Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

    2014-01-01

    The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles. PMID:24694711

  2. Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein.

    PubMed

    Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

    2014-10-01

    The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein-protein or viral-host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.

  3. A genome scan revealed significant associations of growth traits with a major QTL and GHR2 in tilapia

    PubMed Central

    Liu, Feng; Sun, Fei; Xia, Jun Hong; Li, Jian; Fu, Gui Hong; Lin, Grace; Tu, Rong Jian; Wan, Zi Yi; Quek, Delia; Yue, Gen Hua

    2014-01-01

    Growth is an important trait in animal breeding. However, the genetic effects underpinning fish growth variability are still poorly understood. QTL mapping and analysis of candidate genes are effective methods to address this issue. We conducted a genome-wide QTL analysis for growth in tilapia. A total of 10, 7 and 8 significant QTLs were identified for body weight, total length and standard length at 140 dph, respectively. The majority of these QTLs were sex-specific. One major QTL for growth traits was identified in the sex-determining locus in LG1, explaining 71.7%, 67.2% and 64.9% of the phenotypic variation (PV) of body weight, total length and standard length, respectively. In addition, a candidate gene GHR2 in a QTL was significantly associated with body weight, explaining 13.1% of PV. Real-time qPCR revealed that different genotypes at the GHR2 locus influenced the IGF-1 expression level. The markers located in the major QTL for growth traits could be used in marker-assisted selection of tilapia. The associations between GHR2 variants and growth traits suggest that the GHR2 gene should be an important gene that explains the difference in growth among tilapia species. PMID:25435025

  4. Oral treatment with zinc sulfate increases the expression of Th1 cytokines mRNA in BALB/c mice infected with Leishmania major.

    PubMed

    Afshari, Mahsa; Riazi-Rad, Farhad; Khaze, Vahid; Bahrami, Fariborz; Ajdary, Soheila; Alimohammadian, Mohammad Hossein

    2016-05-01

    Leishmaniases consist of a group of diseases caused by protozoan parasites of Leishmania genus. The outcome of the disease depends on the immune responses of the host as well as the pathogenicity of the strain of the parasite. In murine models, the inoculation of Leishmania major into resistant mice results in Th1 responses and recovery from the infection. However in the susceptible mice, the same inoculation leads to a profile of Th2 responses. Zinc (Zn) is an essential trace element which is required for the growth and development of the immune responses. In this study, the influence of Zn sulfate on mRNA expression of main cytokines of the immune response was studied in susceptible BALB/c mice infected with L. major. The inoculated mice were divided into 3 groups, namely the untreated (control), the zinc sulfate treated (weeks 2, 4 and 8), and the Glucantime-treated (weeks 4 and 8) mice. During different time points post-infection, the lesion sizes and the parasite burden were measured in all the groups. Moreover, the expression of Ifng, Il4, Il10 and Il12 mRNA levels in the draining lymph nodes of the treated mice were compared to the control mice using real-time PCR. Our data demonstrated significant decreases in lesion sizes and parasite loads in Zn sulfate treated group compared to the untreated group. Moreover, significant fold increases in expression of Ifng transcript were observed in mice treated with Zn sulfate compared to the control. The ratio of Ifng/Il4 mRNA was also higher in Zn sulfate-treated mice compared to Glucantime-treated animals. These results indicate that Zn Sulfate has the ability to induce strong Th1 responses in susceptible BALB/c mice inoculated with L. major. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Increased myelopoiesis during Leishmania major infection in mice: generation of 'safe targets', a possible way to evade the effector immune mechanism.

    PubMed Central

    Mirkovich, A M; Galelli, A; Allison, A C; Modabber, F Z

    1986-01-01

    BALB/c mice are highly susceptible to Leishmania major infection and develop a disseminated lethal disease. Previous experiments indicate that during infection the spleen is heavily populated with large mononuclear cells containing amastigotes. Morphologically these cells resemble undifferentiated monocytes and granulocytes. In this study we examined myelopoiesis in BALB/c and C57BL/6 (resistant) mice during infection with L. major. The number of macrophage-granulocyte precursors in the spleen of infected BALB/c mice, determined by colony forming units in soft-agar cultures (cfu-c), increased steadily to a level of about 60 times that of normal sex- and age-matched controls. In C57BL/6 mice, spleen cfu-c peaked at about 1 month post-infection (four times that of normal controls) and declined thereafter to about two times normal levels. The number of cfu-c in the bone marrow did not change significantly in either strain during the infection. Colony stimulating activity (CSA) was found in supernates of cultures of adherent cells from the spleen of infected BALB/c mice. Under the same conditions, CSA was non-detectable in supernates of nonadherent spleen cells of infected mice, and those of adherent or nonadherent spleen cells of control animals. A possible role of undifferentiated macrophage-granulocytes in the exquisite susceptibility of BALB/c mice to L. major infection is discussed. PMID:3488146

  6. Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice.

    PubMed

    Cortes-Perez, Naima G; Ah-Leung, Sandrine; Bermúdez-Humarán, Luis G; Corthier, Gérard; Wal, Jean-Michel; Langella, Philippe; Adel-Patient, Karine

    2007-03-01

    The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the

  7. [(18)F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice.

    PubMed

    Kuszpit, Kyle; Hollidge, Bradley S; Zeng, Xiankun; Stafford, Robert G; Daye, Sharon; Zhang, Xiang; Basuli, Falguni; Golden, Joseph W; Swenson, Rolf E; Smith, Darci R; Bocan, Thomas M

    2017-09-12

    The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [(18)F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice. We assessed ZIKV-induced pathogenesis in wild-type C57BL/6 mice administered an antibody to inhibit type I interferon (IFN) signaling. [(18)F]DPA-714 PET imaging was performed on days 3, 6, and 10 post-infection (PI), and tissues were subsequently processed for histological evaluation, quantification of microgliosis, and detection of viral RNA by in situ hybridization (ISH). In susceptible ZIKV-infected mice, viral titers in the brain increased from days 3 to 10 PI. Over this span, these mice showed a two- to sixfold increase in global brain neuroinflammation using [(18)F]DPA-714 PET imaging despite limited, regional detection of viral RNA. No measurable increase in ionized calcium binding adaptor molecule 1 (Iba-1) expression was noted at day 3 PI; however, there was a modest increase at day 6 PI and an approximately significant fourfold increase in Iba-1 expression at day 10 PI in the susceptible ZIKV-infected group relative to controls. The results of the current study demonstrate that global neuroinflammation plays a significant role in the progression of ZIKV infection and that [(18)F]DPA-714 PET imaging is a sensitive tool relative to histology for the detection of neuroinflammation. [(18)F]DPA-714 PET imaging may be useful in dynamically characterizing the pathology associated with neurotropic viruses and the evaluation of therapeutics being developed for treatment of infectious diseases.

  8. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis.

    PubMed

    Zelzer, Elazar; McLean, William; Ng, Yin-Shan; Fukai, Naomi; Reginato, Anthony M; Lovejoy, Stephanie; D'Amore, Patricia A; Olsen, Bjorn R

    2002-04-01

    Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.

  9. T Cells That React to the Immunodominant Leishmania major LACK Antigen Prevent Early Dissemination of the Parasite in Susceptible BALB/c Mice

    PubMed Central

    Schilling, Sabrina; Glaichenhaus, Nicolas

    2001-01-01

    Susceptibility of BALB/c mice to Leishmania major depends on the early production of IL-4 by CD4+ T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination of L. major to the internal organs. PMID:11160025

  10. Phenotypic Characterization of Speed-Associated Gait Changes in Mice Reveals Modular Organization of Locomotor Networks

    PubMed Central

    Bellardita, Carmelo; Kiehn, Ole

    2015-01-01

    SUMMARY Studies of locomotion in mice suggest that circuits controlling the alternating between left and right limbs may have a modular organization with distinct locomotor circuits being recruited at different speeds. It is not clear, however, whether such a modular organization reflects specific behavioral outcomes expressed at different speeds of locomotion. Here, we use detailed kinematic analyses to search for signatures of a modular organization of locomotor circuits in intact and genetically modified mice moving at different speeds of locomotion. We show that wild-type mice display three distinct gaits: two alternating, walk and trot, and one synchronous, bound. Each gait is expressed in distinct ranges of speed with phenotypic inter-limb and intra-limb coordination. A fourth gait, gallop, closely resembled bound in most of the locomotor parameters but expressed diverse inter-limb coordination. Genetic ablation of commissural V0V neurons completely removed the expression of one alternating gait, trot, but left intact walk, gallop, and bound. Ablation of commissural V0V and V0D neurons led to a loss of walk, trot, and gallop, leaving bound as the default gait. Our study provides a benchmark for studies of the neuronal control of locomotion in the full range of speeds. It provides evidence that gait expression depends upon selection of different modules of neuronal ensembles. PMID:25959968

  11. Phenotypic characterization of speed-associated gait changes in mice reveals modular organization of locomotor networks.

    PubMed

    Bellardita, Carmelo; Kiehn, Ole

    2015-06-01

    Studies of locomotion in mice suggest that circuits controlling the alternating between left and right limbs may have a modular organization with distinct locomotor circuits being recruited at different speeds. It is not clear, however, whether such a modular organization reflects specific behavioral outcomes expressed at different speeds of locomotion. Here, we use detailed kinematic analyses to search for signatures of a modular organization of locomotor circuits in intact and genetically modified mice moving at different speeds of locomotion. We show that wild-type mice display three distinct gaits: two alternating, walk and trot, and one synchronous, bound. Each gait is expressed in distinct ranges of speed with phenotypic inter-limb and intra-limb coordination. A fourth gait, gallop, closely resembled bound in most of the locomotor parameters but expressed diverse inter-limb coordination. Genetic ablation of commissural V0V neurons completely removed the expression of one alternating gait, trot, but left intact walk, gallop, and bound. Ablation of commissural V0V and V0D neurons led to a loss of walk, trot, and gallop, leaving bound as the default gait. Our study provides a benchmark for studies of the neuronal control of locomotion in the full range of speeds. It provides evidence that gait expression depends upon selection of different modules of neuronal ensembles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Nerve Growth Factor Promoter Activity Revealed in Mice Expressing Enhanced Green Fluorescent Protein

    PubMed Central

    Kawaja, Michael D.; Smithson, Laura J.; Elliott, Janet; Trinh, Gina; Crotty, Anne-Marie; Michalski, Bernadeta; Fahnestock, Margaret

    2012-01-01

    Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury. PMID:21456011

  13. Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks

    PubMed Central

    Wada, Takeshi; Ishiwata, Kenji; Koseki, Haruhiko; Ishikura, Tomoyuki; Ugajin, Tsukasa; Ohnuma, Naotsugu; Obata, Kazushige; Ishikawa, Ryosuke; Yoshikawa, Soichiro; Mukai, Kaori; Kawano, Yohei; Minegishi, Yoshiyuki; Yokozeki, Hiroo; Watanabe, Naohiro; Karasuyama, Hajime

    2010-01-01

    Ticks are ectoparasitic arthropods that can transmit a variety of microorganisms to humans and animals during blood feeding, causing serious infectious disorders, including Lyme disease. Acaricides are pharmacologic agents that kill ticks. The emergence of acaricide-resistant ticks calls for alternative control strategies for ticks and tick-borne diseases. Many animals develop resistance to ticks after repeated infestations, but the nature of this acquired anti-tick immunity remains poorly understood. Here we investigated the cellular and molecular mechanisms underlying acquired resistance to Haemaphysalis longicornis ticks in mice and found that antibodies were required, as was IgFc receptor expression on basophils but not on mast cells. The infiltration of basophils at tick-feeding sites occurred during the second, but not the first, tick infestation. To assess the requirement for basophil infiltration to acquired tick resistance, mice expressing the human diphtheria toxin receptor under the control of the mast cell protease 8 (Mcpt8) promoter were generated. Diphtheria toxin administration to these mice selectively ablated basophils. Diphtheria toxin–mediated basophil depletion before the second tick infestation resulted in loss of acquired tick resistance. These data provide the first clear evidence, to our knowledge, that basophils play an essential and nonredundant role in antibody-mediated acquired immunity against ticks, which may suggest new strategies for controlling tick-borne diseases. PMID:20664169

  14. 1H NMR-based profiling reveals differential immune-metabolic networks during influenza virus infection in obese mice.

    PubMed

    Milner, J Justin; Wang, Jue; Sheridan, Patricia A; Ebbels, Tim; Beck, Melinda A; Saric, Jasmina

    2014-01-01

    Obese individuals are at greater risk for death from influenza virus infection. Paralleling human evidence, obese mice are also more susceptible to influenza infection mortality. However, the underlying mechanisms driving greater influenza severity in the obese remain unclear. Metabolic profiling has been utilized in infectious disease models to enhance prognostic or diagnostic methods, and to gain insight into disease pathogenesis by providing a more global picture of dynamic infection responses. Herein, metabolic profiling was used to develop a deeper understanding of the complex processes contributing to impaired influenza protection in obese mice and to facilitate generation of new explanatory hypotheses. Diet-induced obese and lean mice were infected with influenza A/Puerto Rico/8/34. 1H nuclear magnetic resonance-based metabolic profiling of urine, feces, lung, liver, mesenteric white adipose tissue, bronchoalveolar lavage fluid and serum revealed distinct metabolic signatures in infected obese mice, including perturbations in nucleotide, vitamin, ketone body, amino acid, carbohydrate, choline and lipid metabolic pathways. Further, metabolic data was integrated with immune analyses to obtain a more comprehensive understanding of potential immune-metabolic interactions. Of interest, uncovered metabolic signatures in urine and feces allowed for discrimination of infection status in both lean and obese mice at an early influenza time point, which holds prognostic and diagnostic implications for this methodology. These results confirm that obesity causes distinct metabolic perturbations during influenza infection and provide a basis for generation of new hypotheses and use of this methodology in detection of putative biomarkers and metabolic patterns to predict influenza infection outcome.

  15. Illumination of Murine Gammaherpesvirus-68 Cycle Reveals a Sexual Transmission Route from Females to Males in Laboratory Mice

    PubMed Central

    François, Sylvie; Vidick, Sarah; Sarlet, Mickaël; Desmecht, Daniel; Drion, Pierre; Stevenson, Philip G.; Vanderplasschen, Alain; Gillet, Laurent

    2013-01-01

    Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these

  16. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

    PubMed Central

    Schroeder, Frank C.; Taggi, Andrew E.; Gronquist, Matthew; Malik, Rabia U.; Grant, Jacqualine B.; Eisner, Thomas; Meinwald, Jerrold

    2008-01-01

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom. PMID:18794518

  17. Genome-Wide Sequencing Reveals Two Major Sub-Lineages in the Genetically Monomorphic Pathogen Xanthomonas Campestris Pathovar Musacearum

    PubMed Central

    Wasukira, Arthur; Tayebwa, Johnbosco; Thwaites, Richard; Paszkiewicz, Konrad; Aritua, Valente; Kubiriba, Jerome; Smith, Julian; Grant, Murray; Studholme, David J.

    2012-01-01

    The bacterium Xanthomonas campestris pathovar musacearum (Xcm) is the causal agent of banana Xanthomonas wilt (BXW). This disease has devastated economies based on banana and plantain crops (Musa species) in East Africa. Here we use genome-wide sequencing to discover a set of single-nucleotide polymorphisms (SNPs) among East African isolates of Xcm. These SNPs have potential as molecular markers for phylogeographic studies of the epidemiology and spread of the pathogen. Our analysis reveals two major sub-lineages of the pathogen, suggesting that the current outbreaks of BXW on Musa species in the region may have more than one introductory event, perhaps from Ethiopia. Also, based on comparisons of genome-wide sequence data from multiple isolates of Xcm and multiple strains of X. vasicola pathovar vasculorum, we identify genes specific to Xcm that could be used to specifically detect Xcm by PCR-based methods. PMID:24704974

  18. Genome-wide sequencing reveals two major sub-lineages in the genetically monomorphic pathogen xanthomonas campestris pathovar musacearum.

    PubMed

    Wasukira, Arthur; Tayebwa, Johnbosco; Thwaites, Richard; Paszkiewicz, Konrad; Aritua, Valente; Kubiriba, Jerome; Smith, Julian; Grant, Murray; Studholme, David J

    2012-07-04

    The bacterium Xanthomonas campestris pathovar musacearum (Xcm) is the causal agent of banana Xanthomonas wilt (BXW). This disease has devastated economies based on banana and plantain crops (Musa species) in East Africa. Here we use genome-wide sequencing to discover a set of single-nucleotide polymorphisms (SNPs) among East African isolates of Xcm. These SNPs have potential as molecular markers for phylogeographic studies of the epidemiology and spread of the pathogen. Our analysis reveals two major sub-lineages of the pathogen, suggesting that the current outbreaks of BXW on Musa species in the region may have more than one introductory event, perhaps from Ethiopia. Also, based on comparisons of genome-wide sequence data from multiple isolates of Xcm and multiple strains of X. vasicola pathovar vasculorum, we identify genes specific to Xcm that could be used to specifically detect Xcm by PCR-based methods.

  19. Characterization of the Early Inflammatory Infiltrate at the Feeding Site of Infected Sand Flies in Mice Protected from Vector-Transmitted Leishmania major by Exposure to Uninfected Bites

    PubMed Central

    Teixeira, Clarissa; Gomes, Regis; Oliveira, Fabiano; Meneses, Claudio; Gilmore, Dana C.; Elnaiem, Dia-Eldin A.; Valenzuela, Jesus G.; Kamhawi, Shaden

    2014-01-01

    Background Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN- γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. Methodology/Principal findings Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. Conclusion/Significance The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection. PMID:24762408

  20. Metabolomics of dates (Phoenix dactylifera) reveals a highly dynamic ripening process accounting for major variation in fruit composition.

    PubMed

    Diboun, Ilhame; Mathew, Sweety; Al-Rayyashi, Maryam; Elrayess, Mohamed; Torres, Maria; Halama, Anna; Méret, Michaël; Mohney, Robert P; Karoly, Edward D; Malek, Joel; Suhre, Karsten

    2015-12-16

    Dates are tropical fruits with appreciable nutritional value. Previous attempts at global metabolic characterization of the date metabolome were constrained by small sample size and limited geographical sampling. In this study, two independent large cohorts of mature dates exhibiting substantial diversity in origin, varieties and fruit processing conditions were measured by metabolomics techniques in order to identify major determinants of the fruit metabolome. Multivariate analysis revealed a first principal component (PC1) significantly associated with the dates' countries of production. The availability of a smaller dataset featuring immature dates from different development stages served to build a model of the ripening process in dates, which helped reveal a strong ripening signature in PC1. Analysis revealed enrichment in the dry type of dates amongst fruits with early ripening profiles at one end of PC1 as oppose to an overrepresentation of the soft type of dates with late ripening profiles at the other end of PC1. Dry dates are typical to the North African region whilst soft dates are more popular in the Gulf region, which partly explains the observed association between PC1 and geography. Analysis of the loading values, expressing metabolite correlation levels with PC1, revealed enrichment patterns of a comprehensive range of metabolite classes along PC1. Three distinct metabolic phases corresponding to known stages of date ripening were observed: An early phase enriched in regulatory hormones, amines and polyamines, energy production, tannins, sucrose and anti-oxidant activity, a second phase with on-going phenylpropanoid secondary metabolism, gene expression and phospholipid metabolism and a late phase with marked sugar dehydration activity and degradation reactions leading to increased volatile synthesis. These data indicate the importance of date ripening as a main driver of variation in the date metabolome responsible for their diverse nutritional and

  1. Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

    PubMed

    Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

    2015-04-03

    Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

  2. A genome-wide scan for human obesity genes reveals a major susceptibility locus on chromosome 10.

    PubMed

    Hager, J; Dina, C; Francke, S; Dubois, S; Houari, M; Vatin, V; Vaillant, E; Lorentz, N; Basdevant, A; Clement, K; Guy-Grand, B; Froguel, P

    1998-11-01

    Obesity, a common multifactorial disorder, is a major risk factor for type 2 diabetes, hypertension and coronary heart disease (CHD). According to the definition of the World Health Organization (WHO), approximately 6-10% of the population in Westernized countries are considered obese. Epidemiological studies have shown that 30-70% of the variation in body weight may be attributable to genetic factors. To date, two genome-wide scans using different obesity-related quantitative traits have provided candidate regions for obesity. We have undertaken a genome-wide scan in affected sibpairs to identify chromosomal regions linked to obesity in a collection of French families. Model-free multipoint linkage analyses revealed evidence for linkage to a region on chromosome 10p (MLS=4.85). Two further loci on chromosomes 5cen-q and 2p showed suggestive evidence for linkage of serum leptin levels in a genome-wide context. The peak on chromosome 2 coincided with the region containing the gene (POMC) encoding pro-opiomelanocortin, a locus previously linked to leptin levels and fat mass in a Mexican-American population and shown to be mutated in obese humans. Our results suggest that there is a major gene on chromosome 10p implicated in the development of human obesity, and the existence of two further loci influencing leptin levels.

  3. Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants.

    PubMed

    Robertson, D; Mitchell, G P; Gilroy, J S; Gerrish, C; Bolwell, G P; Slabas, A R

    1997-06-20

    The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.

  4. Genetic Analysis Reveals That C19-GA 2-Oxidation Is a Major Gibberellin Inactivation Pathway in Arabidopsis[W

    PubMed Central

    Rieu, Ivo; Eriksson, Sven; Powers, Stephen J.; Gong, Fan; Griffiths, Jayne; Woolley, Lindsey; Benlloch, Reyes; Nilsson, Ove; Thomas, Stephen G.; Hedden, Peter; Phillips, Andrew L.

    2008-01-01

    Bioactive hormone concentrations are regulated both at the level of hormone synthesis and through controlled inactivation. Based on the ubiquitous presence of 2β-hydroxylated gibberellins (GAs), a major inactivating pathway for the plant hormone GA seems to be via GA 2-oxidation. In this study, we used various approaches to determine the role of C19-GA 2-oxidation in regulating GA concentration and GA-responsive plant growth and development. We show that Arabidopsis thaliana has five C19-GA 2-oxidases, transcripts for one or more of which are present in all organs and at all stages of development examined. Expression of four of the five genes is subject to feed-forward regulation. By knocking out all five Arabidopsis C19-GA 2-oxidases, we show that C19-GA 2-oxidation limits bioactive GA content and regulates plant development at various stages during the plant life cycle: C19-GA 2-oxidases prevent seed germination in the absence of light and cold stimuli, delay the vegetative and floral phase transitions, limit the number of flowers produced per inflorescence, and suppress elongation of the pistil prior to fertilization. Under GA-limited conditions, further roles are revealed, such as limiting elongation of the main stem and side shoots. We conclude that C19-GA 2-oxidation is a major GA inactivation pathway regulating development in Arabidopsis. PMID:18805991

  5. Speed-Dependent Modulation of the Locomotor Behavior in Adult Mice Reveals Attractor and Transitional Gaits

    PubMed Central

    Lemieux, Maxime; Josset, Nicolas; Roussel, Marie; Couraud, Sébastien; Bretzner, Frédéric

    2016-01-01

    Locomotion results from an interplay between biomechanical constraints of the muscles attached to the skeleton and the neuronal circuits controlling and coordinating muscle activities. Quadrupeds exhibit a wide range of locomotor gaits. Given our advances in the genetic identification of spinal and supraspinal circuits important to locomotion in the mouse, it is now important to get a better understanding of the full repertoire of gaits in the freely walking mouse. To assess this range, young adult C57BL/6J mice were trained to walk and run on a treadmill at different locomotor speeds. Instead of using the classical paradigm defining gaits according to their footfall pattern, we combined the inter-limb coupling and the duty cycle of the stance phase, thus identifying several types of gaits: lateral walk, trot, out-of-phase walk, rotary gallop, transverse gallop, hop, half-bound, and full-bound. Out-of-phase walk, trot, and full-bound were robust and appeared to function as attractor gaits (i.e., a state to which the network flows and stabilizes) at low, intermediate, and high speeds respectively. In contrast, lateral walk, hop, transverse gallop, rotary gallop, and half-bound were more transient and therefore considered transitional gaits (i.e., a labile state of the network from which it flows to the attractor state). Surprisingly, lateral walk was less frequently observed. Using graph analysis, we demonstrated that transitions between gaits were predictable, not random. In summary, the wild-type mouse exhibits a wider repertoire of locomotor gaits than expected. Future locomotor studies should benefit from this paradigm in assessing transgenic mice or wild-type mice with neurotraumatic injury or neurodegenerative disease affecting gait. PMID:26941592

  6. Task-specific enhancement of hippocampus-dependent learning in mice deficient in monoacylglycerol lipase, the major hydrolyzing enzyme of the endocannabinoid 2-arachidonoylglycerol

    PubMed Central

    Kishimoto, Yasushi; Cagniard, Barbara; Yamazaki, Maya; Nakayama, Junko; Sakimura, Kenji; Kirino, Yutaka; Kano, Masanobu

    2015-01-01

    Growing evidence indicates that the endocannabinoid system is important for the acquisition and/or extinction of learning and memory. However, it is unclear which endocannabinoid(s) play(s) a crucial role in these cognitive functions, especially memory extinction. To elucidate the physiological role of 2-arachidonoylglycerol (2-AG), a major endocannabinoid, in behavioral and cognitive functions, we conducted a comprehensive behavioral test battery in knockout (KO) mice deficient in monoacylglycerol lipase (MGL), the major hydrolyzing enzyme of 2-AG. We found age-dependent increases in spontaneous physical activity (SPA) in MGL KO mice. Next, we tested the MGL KO mice using 5 hippocampus-dependent learning paradigms (i.e., Morris water maze (MWM), contextual fear conditioning, novel object recognition test, trace eyeblink conditioning, and water-finding test). In the MWM, MGL KO mice showed normal acquisition of reference memory, but exhibited significantly faster extinction of the learned behavior. Moreover, they showed faster memory acquisition on the reversal-learning task of the MWM. In contrast, in the contextual fear conditioning, MGL KO mice tended to show slower memory extinction. In the novel object recognition and water-finding tests, MGL KO mice exhibited enhanced memory acquisition. Trace eyeblink conditioning was not altered in MGL KO mice throughout the acquisition and extinction phases. These results indicate that 2-AG signaling is important for hippocampus-dependent learning and memory, but its contribution is highly task-dependent. PMID:26082696

  7. Depletion of interleukin-4 in BALB/c mice with established Leishmania major infections increases the efficacy of antimony therapy and promotes Th1-like responses.

    PubMed Central

    Nabors, G S; Farrell, J P

    1994-01-01

    Whereas most inbred mouse strains mount a protective Th1 helper T-cell response following infection with Leishmania major, an ineffective Th2 response develops in BALB/c mice, leading to the development of disseminated, ultimately fatal disease. Interleukin-4 (IL-4) production is required for the initiation of the Th2 response, though little is known about the requirements for the long-term maintenance of this response. In order to investigate the role of the expanding parasite population on the Th2 response, mice infected for 2 weeks with L. major, which exhibited a Th2-like cytokine profile, were treated with a leishmanicidal agent (Pentostam) and/or various doses of anti-IL-4 antibody. Untreated mice, mice treated with Pentostam alone, or mice treated with 2.5 mg of anti-IL-4 antibody given at days 13 and 21 of infection developed progressive disease. However, in 8 of 10 mice treated with this dose of anti-IL-4 antibody plus Pentostam lesion development was arrested and lesions were either controlled or eventually healed. Healing was associated with the production of high levels of gamma interferon by spleen cells, and low levels of immunoglobulin E in serum compared with levels for control animals, indicating that a Th1-like response had developed in mice receiving both treatments. Thus, depletion of IL-4 only in combination with a reduction in the parasite burden allowed the expression of a Th1 response. When the dose of anti-IL-4 antibody was increased to 5 mg per injection, all mice treated with this dose of antibody, with or without Pentostam therapy, healed. However, combined therapy with Pentostam in mice treated with this dose of antibody had an additional protective effect. As expected, a Th1 response developed in mice treated with this dose of anti-IL-4 antibody with or without combined therapy with Pentostam, whereas a Th2 response developed in control mice. Thus, a significant effect on the course of disease is noted when mice with established L

  8. Colony-stimulating factor-1 plays a major role in the development of reproductive function in male mice.

    PubMed

    Cohen, P E; Hardy, M P; Pollard, J W

    1997-10-01

    Colony-stimulating factor-1 (CSF-1) is the principal regulator of cells of the mononuclear phagocytic lineage that includes monocytes, tissue macrophages, microglia, and osteoclasts. Macrophages are found throughout the reproductive tract of both males and females and have been proposed to act as regulators of fertility at several levels. Mice homozygous for the osteopetrosis mutation (csfm[op]) lack CSF-1 and, consequently, have depleted macrophage numbers. Further analysis has revealed that male csfm(op)/csfm(op) mice have reduced mating ability, low sperm numbers, and 90% lower serum testosterone levels. The present studies show that this low serum testosterone is due to reduced testicular Leydig cell steroidogenesis associated with severe ultrastructural abnormalities characterized by disrupted intracellular membrane structures. In addition, the Leydig cells from csfm(op)/ csfm(op) males have diminished amounts of the steroidogenic enzyme proteins P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and P450 17alpha-hydroxylase-lyase, with associated reductions in the activity of all these steroidogenic enzymes, as well as in 17beta-hydroxysteroid dehydrogenase. The CSF-1-deficient males also have reduced serum LH and disruption of the normal testosterone negative feedback response of the hypothalamus, as demonstrated by the failure to increase LH secretion in castrated males and their lack of response to exogenous testosterone. However, these males are responsive to GnRH and LH treatment. These studies have identified a novel role for CSF-1 in the development and/or regulation of the male hypothalamic-pituitary-gonadal axis.

  9. Characterization of connexin31.1-deficient mice reveals impaired placental development.

    PubMed

    Zheng-Fischhöfer, Qingyi; Kibschull, Mark; Schnichels, Marc; Kretz, Markus; Petrasch-Parwez, Elisabeth; Strotmann, Jörg; Reucher, Harald; Lynn, Bruce D; Nagy, James I; Lye, Stephen J; Winterhager, Elke; Willecke, Klaus

    2007-12-01

    The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using beta-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.

  10. Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency.

    PubMed

    Weng, Rui; Shen, Sensen; Burton, Casey; Yang, Li; Nie, Honggang; Tian, Yonglu; Bai, Yu; Liu, Huwei

    2016-04-01

    Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2-/-) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2-/- mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.

  11. Caloric restriction reveals a metabolomic and lipidomic signature in liver of male mice.

    PubMed

    Jové, Mariona; Naudí, Alba; Ramírez-Núñez, Omar; Portero-Otín, Manuel; Selman, Colin; Withers, Dominic J; Pamplona, Reinald

    2014-10-01

    Lipid composition, particularly membrane unsaturation, has been proposed as being a lifespan determinant, but it is currently unknown whether caloric restriction (CR), an accepted life-extending intervention, affects cellular lipid profiles. In this study, we employ a liquid chromatography quadrupole time-of-flight-based methodology to demonstrate that CR in the liver of male C57BL/6 mice: (i) induces marked changes in the cellular lipidome, (ii) specifically reduces levels of a phospholipid peroxidation product, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphatidylcholine, (iii) alters cellular phosphoethanolamine and triglyceride distributional profiles, (iv) affects mitochondrial electron transport chain complexes, increasing complex II and decreasing complex III and (v) is associated with specific changes in liver metabolic pathways. These data demonstrate that CR induces a specific lipidome and metabolome reprogramming event in mouse liver which is associated with lower protein oxidative damage, as assessed by mass spectrometry-based measurements. Such changes may be critical to the increased lifespan and healthspan observed in C57BL/6 mice following CR.

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

    PubMed Central

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

    2010-01-01

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

  13. In vivo HMRS and lipidomic profiling reveals comprehensive changes of hippocampal metabolism during aging in mice.

    PubMed

    Lin, Lejun; Cao, Bofeng; Xu, Zhiying; Sui, Yanbin; Chen, Jiao; Luan, Qiang; Yang, Ruifang; Li, Shanchun; Li, Ke Feng

    2016-01-29

    Aging is characterized by various cellular changes in the brain. Hippocampus is important for systemic aging and lifespan control. There is still a lack of comprehensive overview of metabolic changes in hippocampus during aging. In this study, we first created an accelerated brain aging mice model through the chronic administration of d-galactose. We then performed a multiplatform metabolomic profiling of mice hippocampus using the combination of in vivo 9.4 T HMRS and in vitro LC-MS/MS based lipidomics. We found N-acetylaspartic acid (NAA), gama-aminobutyric acid (GABA), glutamate/glutamine, taurine, choline, sphingolipids (SMs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs) and phosphatidylserines (PSs), all of them decreasing with the aging process in mice hippocampus. The changes of sphingolipids and phospholipids were not limited to one single class or molecular species. In contrast, we found the significant accumulation of lactate, myoinositol and phosphatidylcholines (PCs) along with aging in hippocampus. SM (d18:1/20:2), PE (36:2), PG (34:1), PI (36:4), PS (18:0/20:4) and PC (36:0) have the most significant changes along with aging. Network analysis revealed the striking loss of biochemical connectivity and interactions between hippocampal metabolites with aging. The correlation pattern between metabolites in hippocampus could function as biomarkers for aging or diagnosis of aging-related diseases.

  14. Precise genetic mapping and integrative bioinformatics in Diversity Outbred mice reveals Hydin as a novel pain gene.

    PubMed

    Recla, Jill M; Robledo, Raymond F; Gatti, Daniel M; Bult, Carol J; Churchill, Gary A; Chesler, Elissa J

    2014-06-01

    Mouse genetics is a powerful approach for discovering genes and other genome features influencing human pain sensitivity. Genetic mapping studies have historically been limited by low mapping resolution of conventional mouse crosses, resulting in pain-related quantitative trait loci (QTL) spanning several megabases and containing hundreds of candidate genes. The recently developed Diversity Outbred (DO) population is derived from the same eight inbred founder strains as the Collaborative Cross, including three wild-derived strains. DO mice offer increased genetic heterozygosity and allelic diversity compared to crosses involving standard mouse strains. The high rate of recombinatorial precision afforded by DO mice makes them an ideal resource for high-resolution genetic mapping, allowing the circumvention of costly fine-mapping studies. We utilized a cohort of ~300 DO mice to map a 3.8 Mbp QTL on chromosome 8 associated with acute thermal pain sensitivity, which we have tentatively named Tpnr6. We used haplotype block partitioning to narrow Tpnr6 to a width of ~230 Kbp, reducing the number of putative candidate genes from 44 to 3. The plausibility of each candidate gene's role in pain response was assessed using an integrative bioinformatics approach, combining data related to protein domain, biological annotation, gene expression pattern, and protein functional interaction. Our results reveal a novel, putative role for the protein-coding gene, Hydin, in thermal pain response, possibly through the gene's role in ciliary motility in the choroid plexus-cerebrospinal fluid system of the brain. Real-time quantitative-PCR analysis showed no expression differences in Hydin transcript levels between pain-sensitive and pain-resistant mice, suggesting that Hydin may influence hot-plate behavior through other biological mechanisms.

  15. Lipidomics Analysis Reveals Efficient Storage of Hepatic Triacylglycerides Enriched in Unsaturated Fatty Acids after One Bout of Exercise in Mice

    PubMed Central

    Zhao, Xinjie; Häring, Hans U.; Schleicher, Erwin; Lehmann, Rainer; Han, Xianlin; Xu, Guowang; Weigert, Cora

    2010-01-01

    Background Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise. Methodology/Principal Findings We performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG. Conclusion These data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation. PMID

  16. Gene for ovarian granulosa cell tumor susceptibility, Gct, in SWXJ recombinant inbred strains of mice revealed by dehydroepiandrosterone.

    PubMed

    Beamer, W G; Tennent, B J; Shultz, K L; Nadeau, J H; Shultz, L D; Skow, L C

    1988-09-15

    Spontaneous, malignant ovarian granulosa cell (GC) tumors occur in pubertal SWR and specific SWXJ recombinant inbred strains of mice. Treatment of these mice with dehydroepiandrosterone (DHEA), an adrenal secretory steroid with anticancer actions against spontaneous and carcinogen-induced tumors of different tissues, gave unexpected results. Diet supplemented with 0.4% DHEA (a) induced significantly more GC tumors in spontaneous tumor-susceptible strains (SWR and SWXJ-1, -4, and -9), (b) induced the first GC tumors observed in five previously tumor-free strains (SWXJ-6, -7, -8, -10, and -12), and (c) failed to induce GC tumors in SJL and in the remaining six SWXJ strains (SWXJ-2, -3, -5, -11, -13, and -14). The strain distribution pattern of DHEA-induced GC tumor susceptibility versus resistance was compared with strain distribution patterns for 35 different loci known to distinguish SWR and SJL progenitor strains. A complete match of DHEA-induced GC tumors with pancreas-2 (Pan-2) on mouse chromosome 4 was found. We have named this new locus GC tumor susceptibility (Gct), with the Gcts (susceptible) allele found in SWR and the Gctr (resistant) allele found in SJL mice. The Gct locus is closely linked to pancreas-2, Pan-2, but the order of genes is not yet confirmed. In addition, data from F1 progeny of matings between SWR and selected inbred strains provide suggestive evidence for a second gene controlling GC tumor incidence that we hypothesize involves steroid metabolism. Differences in GC tumor incidence data from reciprocal F1 progeny of matings between SWR and SJL mice reveal a strong maternal effect that may represent yet a third gene. These data support a heritable basis for GC tumorigenesis in the SWR model involving a small number of genes.

  17. In vivo cell-autonomous transcriptional abnormalities revealed in mice expressing mutant huntingtin in striatal but not cortical neurons.

    PubMed

    Thomas, Elizabeth A; Coppola, Giovanni; Tang, Bin; Kuhn, Alexandre; Kim, SoongHo; Geschwind, Daniel H; Brown, Timothy B; Luthi-Carter, Ruth; Ehrlich, Michelle E

    2011-03-15

    Huntington's disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared with those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support. While genes related to excitotoxicity, dopamine signaling and trophic support were altered in both DE5 and R6/2 mice, which may be either cell autonomous or non-cell autonomous, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell-autonomous mechanisms. Overall, HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt, in the absence of expression in cortical neurons.

  18. Effect of hydroalcoholic extract of Echinacea purpurea in combination with meglumine antimoniate on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/c mice

    PubMed Central

    Sarkari, Bahador; Mohseni, Mobin; Moein, Mahmoud Reza; Shahriarirad, Reza; Asgari, Qasem

    2017-01-01

    Context: Progressive resistance of Leishmania parasite to available drugs including, meglumine antimoniate, has been reported from various regions of the world, especially Iran. Aims: This study was conducted to evaluate the effect of hydroalcoholic extract of Echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by Leishmania major. Materials and Methods: Hydroalcoholic extract of E. purpurea was prepared from the plant. Amastigote form of L. major was inoculated to the tail base of thirty mice. After their tails became wounded, mice were divided into six groups. The first group was used as control and the second group received 100 mg/kg of Echinacea extract (orally). The third group was treated by meglumine antimoniate with dose of 20 mg/kg. Combination therapy was used for group four, five, and six where the mice received a different concentration of extract (100–200 mg/kg) and glucantime (10–20 mg/kg). The size of the cutaneous lesion on tail base was measured regularly. Findings were analyzed by SPSS software and using Kruskal-Wallis test. Results: The sizes of the lesion were increased in all mice of control group by the time. The mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice, but the differences were not statistically significant (P > 0.05). On the other hand, the differences between the group of mice which received meglumine antimoniate alone, and the rest of groups were statistically significant (P < 0.05). Conclusion: E. purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against L. major in BALB/C mice. PMID:28251109

  19. Effect of hydroalcoholic extract of Echinacea purpurea in combination with meglumine antimoniate on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/c mice.

    PubMed

    Sarkari, Bahador; Mohseni, Mobin; Moein, Mahmoud Reza; Shahriarirad, Reza; Asgari, Qasem

    2017-01-01

    Progressive resistance of Leishmania parasite to available drugs including, meglumine antimoniate, has been reported from various regions of the world, especially Iran. This study was conducted to evaluate the effect of hydroalcoholic extract of Echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by Leishmania major. Hydroalcoholic extract of E. purpurea was prepared from the plant. Amastigote form of L. major was inoculated to the tail base of thirty mice. After their tails became wounded, mice were divided into six groups. The first group was used as control and the second group received 100 mg/kg of Echinacea extract (orally). The third group was treated by meglumine antimoniate with dose of 20 mg/kg. Combination therapy was used for group four, five, and six where the mice received a different concentration of extract (100-200 mg/kg) and glucantime (10-20 mg/kg). The size of the cutaneous lesion on tail base was measured regularly. Findings were analyzed by SPSS software and using Kruskal-Wallis test. The sizes of the lesion were increased in all mice of control group by the time. The mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice, but the differences were not statistically significant (P > 0.05). On the other hand, the differences between the group of mice which received meglumine antimoniate alone, and the rest of groups were statistically significant (P < 0.05). E. purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against L. major in BALB/C mice.

  20. Immunogenomics reveal molecular circuits of diclofenac induced liver injury in mice

    PubMed Central

    Lee, Eun-Hee; Oh, Jung-Hwa; Selvaraj, Saravanakumar; Park, Se-Myo; Choi, Mi-Sun; Spanel, Reinhard

    2016-01-01

    Diclofenac is a non-steroidal anti-inflammatory drug and its use can be associated with severe adverse reactions, notably myocardial infarction, stroke and drug-induced liver injury (DILI). In pursue of immune-mediated DILI mechanisms an immunogenomic study was carried out. Diclofenac treatment of mice at 30 mg/kg for 3 days caused significant serum ALT and AST elevations, hepatomegaly and degenerative changes including hepatic glycogen depletion, hydropic swelling, cholesterolosis and eosinophilic hepatocytes with one animal presenting subsegmental infarction due to portal vein thrombosis. Furthermore, portal/periportal induction of the rate limiting enzyme in ammonia detoxification, i.e. carbamoyl phosphate synthetase 1 was observed. The performed microarray studies informed on > 600 differential expressed genes of which 35, 37 and 50 coded for inflammation, 51, 44 and 61 for immune and 116, 129 and 169 for stress response, respectively after single and repeated dosing for 3 and 14 days. Bioinformatic analysis defined molecular circuits of hepatic inflammation with the growth hormone (Ghr)− and leptin receptor, the protein-tyrosine-phosphatase, selectin and the suppressor-of-cytokine-signaling (Socs) to function as key nodes in gene regulatory networks. Western blotting confirmed induction of fibronectin and M-CSF to hallmark tissue repair and differentiation of monocytes and macrophages. Transcript expression of the macrophage receptor with collagenous structure increased > 7-fold and immunohistochemistry of CD68 evidenced activation of tissue-resident macrophages. Importantly, diclofenac treatment prompted strong expression of phosphorylated Stat3 amongst individual animals and the associated 8- and 4-fold Soc3 and Il-6 induction reinforced Ghr degradation as evidenced by immunoblotting. Moreover, immunohistochemistry confirmed regulation of master regulatory proteins of diclofenac treated mice to suggest complex pro-and anti-inflammatory reactions in immune

  1. Quantitative analysis of light-phase restricted feeding reveals metabolic dyssynchrony in mice.

    PubMed

    Bray, M S; Ratcliffe, W F; Grenett, M H; Brewer, R A; Gamble, K L; Young, M E

    2013-06-01

    Considerable evidence suggests that the time of day at which calories are consumed markedly impacts body weight gain and adiposity. However, a precise quantification of energy balance parameters during controlled animal studies enforcing time-of-day-restricted feeding is currently lacking in the absence of direct human interaction. The purpose of the present study was therefore to quantify the effects of restricted feeding during the light (sleep)-phase in a fully-automated, computer-controlled comprehensive laboratory animal monitoring system (CLAMS) designed to modulate food access in a time-of-day-dependent manner. Energy balance, gene expression (within metabolically relevant tissues), humoral factors and body weight were assessed. We report that relative to mice fed only during the dark (active)-phase, light (sleep)-phase fed mice: (1) consume a large meal upon initiation of food availability; (2) consume greater total calories per day; (3) exhibit a higher respiratory exchange ratio (indicative of decreased reliance on lipid/fatty acid oxidation); (4) exhibit tissue-specific alterations in the phases and amplitudes of circadian clock and metabolic genes in metabolically active tissues (greatest phase differences observed in the liver and diminution of amplitudes in epididymal fat, gastrocnemius muscle and heart); (5) exhibit diminished amplitude in humoral factor diurnal variations (for example, corticosterone); and (6) exhibit greater weight gain within 9 days of restricted feeding. Collectively, these data suggest that weight gain following light (sleep)-phase restricted feeding is associated with significant alterations in energy balance, as well as dyssynchrony between metabolically active organs.

  2. Functional principal component analysis reveals discriminating categories of retinal pigment epithelial morphology in mice.

    PubMed

    Jiang, Yi; Qi, Xin; Chrenek, Micah A; Gardner, Christopher; Boatright, Jeffrey H; Grossniklaus, Hans E; Nickerson, John M

    2013-11-05

    To determine whether multivariate, functional principal component analysis of the size and shape of retinal pigment epithelial (RPE) cell morphology allows discrimination of mouse RPE genotypes and age. Flatmounts of RPE sheets obtained from C57BL/6J (n = 50) and rd10 (n = 61) mice at postnatal days 30 to 720 were stained for zonula occludens-1 (ZO-1) and imaged with confocal microscopy. A total of 111 flatmounts were prepared. Twenty-one morphometric measurements were made on tiled, composite images of complete flatmounts, including cell location, area, and eccentricity, using automated image analysis software for quantitatively measuring cell phenotypes. In young (≤61-day-old) C57BL/6J mice, the RPE morphology resembled a regular hexagonal array of cells of uniform size throughout the retina, except near the ciliary body, where the shapes of RPE resembled a soft network. Old (≥180-day-old) C57BL/6J eyes had a subpopulation of large cells. A clear disruption of the regular cell size and shape appeared in rd10 mutants. Aspect ratio and cell area gave rise to principal components that predictively classified mouse age and genotype. Quantitative differences in the RPE sheet morphology allowed discrimination of rd10 from C57BL/6J strains despite the confounding effect of aging. This has implications for RPE sheet morphology as a potential early biomarker for diagnosis of eye disease and prognosis of the eye at early stages when disease is subtle. We conclude that an RPE cell's area and aspect ratio are very early quantitative indicators that predict progression to advanced RPE disease as manifested in rd10.

  3. Malaria protection in β2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including γδT cells

    PubMed Central

    Taniguchi, Tomoyo; Tachikawa, Saoko; Kanda, Yasuhiro; Kawamura, Toshihiko; Tomiyama-Miyaji, Chikako; Li, Changchun; Watanabe, Hisami; Sekikawa, Hiroho; Abo, Toru

    2007-01-01

    It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in β2-microglobulin-deficient (β2m(–/–)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8+ T cells and natural killer T (NKT) cells. When C57BL/6 and β2m(–/–) mice were injected with parasitized (Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1– αβΤ-cell receptorint cells in both mice. The difference was a compensatory expansion of NK and γδT cells in β2m(–/–) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8+ T and NKT cells may be partially related to this protection. PMID:17916163

  4. The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge.

    PubMed

    Pal, Sukumar; Tifrea, Delia F; Follmann, Frank; Andersen, Peter; de la Maza, Luis M

    2017-03-23

    Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10(4) inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-γ and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost significantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were significantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had significantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were significantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.

  5. Genome Alignment Spanning Major Poaceae Lineages Reveals Heterogeneous Evolutionary Rates and Alters Inferred Dates for Key Evolutionary Events.

    PubMed

    Wang, Xiyin; Wang, Jingpeng; Jin, Dianchuan; Guo, Hui; Lee, Tae-Ho; Liu, Tao; Paterson, Andrew H

    2015-06-01

    Multiple comparisons among genomes can clarify their evolution, speciation, and functional innovations. To date, the genome sequences of eight grasses representing the most economically important Poaceae (grass) clades have been published, and their genomic-level comparison is an essential foundation for evolutionary, functional, and translational research. Using a formal and conservative approach, we aligned these genomes. Direct comparison of paralogous gene pairs all duplicated simultaneously reveal striking variation in evolutionary rates among whole genomes, with nucleotide substitution slowest in rice and up to 48% faster in other grasses, adding a new dimension to the value of rice as a grass model. We reconstructed ancestral genome contents for major evolutionary nodes, potentially contributing to understanding the divergence and speciation of grasses. Recent fossil evidence suggests revisions of the estimated dates of key evolutionary events, implying that the pan-grass polyploidization occurred ∼96 million years ago and could not be related to the Cretaceous-Tertiary mass extinction as previously inferred. Adjusted dating to reflect both updated fossil evidence and lineage-specific evolutionary rates suggested that maize subgenome divergence and maize-sorghum divergence were virtually simultaneous, a coincidence that would be explained if polyploidization directly contributed to speciation. This work lays a solid foundation for Poaceae translational genomics. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  6. Association mapping reveals the genetic architecture of tomato response to water deficit: focus on major fruit quality traits

    PubMed Central

    Albert, Elise; Segura, Vincent; Gricourt, Justine; Bonnefoi, Julien; Derivot, Laurent; Causse, Mathilde

    2016-01-01

    Water scarcity constitutes a crucial constraint for agriculture productivity. High-throughput approaches in model plant species identified hundreds of genes potentially involved in survival under drought, but few having beneficial effects on quality and yield. Nonetheless, controlled water deficit may improve fruit quality through higher concentration of flavor compounds. The underlying genetic determinants are still poorly known. In this study, we phenotyped 141 highly diverse small fruit tomato accessions for 27 traits under two contrasting watering conditions. A subset of 55 accessions exhibited increased metabolite contents and maintained yield under water deficit. Using 6100 single nucleotide polymorphisms (SNPs), association mapping revealed 31, 41, and 44 quantitative trait loci (QTLs) under drought, control, and both conditions, respectively. Twenty-five additional QTLs were interactive between conditions, emphasizing the interest in accounting for QTLs by watering regime interactions in fruit quality improvement. Combining our results with the loci previously identified in a biparental progeny resulted in 11 common QTLs and contributed to a first detailed characterization of the genetic determinants of response to water deficit in tomato. Major QTLs for fruit quality traits were dissected and candidate genes were proposed using expression and polymorphism data. The outcomes provide a basis for fruit quality improvement under deficit irrigation while limiting yield losses. PMID:27856709

  7. Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous-Paleogene boundary.

    PubMed

    Feng, Yan-Jie; Blackburn, David C; Liang, Dan; Hillis, David M; Wake, David B; Cannatella, David C; Zhang, Peng

    2017-07-18

    Frogs (Anura) are one of the most diverse groups of vertebrates and comprise nearly 90% of living amphibian species. Their worldwide distribution and diverse biology make them well-suited for assessing fundamental questions in evolution, ecology, and conservation. However, despite their scientific importance, the evolutionary history and tempo of frog diversification remain poorly understood. By using a molecular dataset of unprecedented size, including 88-kb characters from 95 nuclear genes of 156 frog species, in conjunction with 20 fossil-based calibrations, our analyses result in the most strongly supported phylogeny of all major frog lineages and provide a timescale of frog evolution that suggests much younger divergence times than suggested by earlier studies. Unexpectedly, our divergence-time analyses show that three species-rich clades (Hyloidea, Microhylidae, and Natatanura), which together comprise ∼88% of extant anuran species, simultaneously underwent rapid diversification at the Cretaceous-Paleogene (K-Pg) boundary (KPB). Moreover, anuran families and subfamilies containing arboreal species originated near or after the KPB. These results suggest that the K-Pg mass extinction may have triggered explosive radiations of frogs by creating new ecological opportunities. This phylogeny also reveals relationships such as Microhylidae being sister to all other ranoid frogs and African continental lineages of Natatanura forming a clade that is sister to a clade of Eurasian, Indian, Melanesian, and Malagasy lineages. Biogeographical analyses suggest that the ancestral area of modern frogs was Africa, and their current distribution is largely associated with the breakup of Pangaea and subsequent Gondwanan fragmentation.

  8. Association mapping reveals the genetic architecture of tomato response to water deficit: focus on major fruit quality traits.

    PubMed

    Albert, Elise; Segura, Vincent; Gricourt, Justine; Bonnefoi, Julien; Derivot, Laurent; Causse, Mathilde

    2016-12-01

    Water scarcity constitutes a crucial constraint for agriculture productivity. High-throughput approaches in model plant species identified hundreds of genes potentially involved in survival under drought, but few having beneficial effects on quality and yield. Nonetheless, controlled water deficit may improve fruit quality through higher concentration of flavor compounds. The underlying genetic determinants are still poorly known. In this study, we phenotyped 141 highly diverse small fruit tomato accessions for 27 traits under two contrasting watering conditions. A subset of 55 accessions exhibited increased metabolite contents and maintained yield under water deficit. Using 6100 single nucleotide polymorphisms (SNPs), association mapping revealed 31, 41, and 44 quantitative trait loci (QTLs) under drought, control, and both conditions, respectively. Twenty-five additional QTLs were interactive between conditions, emphasizing the interest in accounting for QTLs by watering regime interactions in fruit quality improvement. Combining our results with the loci previously identified in a biparental progeny resulted in 11 common QTLs and contributed to a first detailed characterization of the genetic determinants of response to water deficit in tomato. Major QTLs for fruit quality traits were dissected and candidate genes were proposed using expression and polymorphism data. The outcomes provide a basis for fruit quality improvement under deficit irrigation while limiting yield losses.

  9. SNP Profile within the Human Major Histocompatibility Complex Reveals an Extreme and Interrupted Level of Nucleotide Diversity

    PubMed Central

    Gaudieri, Silvana; Dawkins, Roger L.; Habara, Kaori; Kulski, Jerzy K.; Gojobori, Takashi

    2000-01-01

    The human major histocompatibility complex (MHC) is characterized by polymorphic multicopy gene families, such as HLA and MIC (PERB11); duplications; insertions and deletions (indels); and uneven rates of recombination. Polymorphisms at the antigen recognition sites of the HLA class I and II genes and at associated neutral sites have been attributed to balancing selection and a hitchhiking effect, respectively. We, and others, have previously shown that nucleotide diversity between MHC haplotypes at non-HLA sites is unusually high (>10%) and up to several times greater than elsewhere in the genome (0.08%–0.2%). We report here the most extensive analysis of nucleotide diversity within a continuous sequence in the genome. We constructed a single nucleotide polymorphism (SNP) profile that reveals a pattern of extreme but interrupted levels of nucleotide diversity by comparing a continuous sequence within haplotypes in three genomic subregions of the MHC. A comparison of several haplotypes within one of the genomic subregions containing the HLA-B and -C loci suggests that positive selection is operating over the whole subgenomic region, including HLA and non-HLA genes. [The sequence data for the multiple haplotype comparisons within the class I region have been submitted to DDBJ/EMBL/GenBank under accession nos. AF029061, AF029062, and AB031005–AB031010. Additional sequence data have been submitted to the DDBJ data library under accession nos. AB031005–AB03101 and AF029061–AF029062.] PMID:11042155

  10. A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification

    PubMed Central

    Macqueen, Daniel J.; Johnston, Ian A.

    2014-01-01

    Whole genome duplication (WGD) is often considered to be mechanistically associated with species diversification. Such ideas have been anecdotally attached to a WGD at the stem of the salmonid fish family, but remain untested. Here, we characterized an extensive set of gene paralogues retained from the salmonid WGD, in species covering the major lineages (subfamilies Salmoninae, Thymallinae and Coregoninae). By combining the data in calibrated relaxed molecular clock analyses, we provide the first well-constrained and direct estimate for the timing of the salmonid WGD. Our results suggest that the event occurred no later in time than 88 Ma and that 40–50 Myr passed subsequently until the subfamilies diverged. We also recovered a Thymallinae–Coregoninae sister relationship with maximal support. Comparative phylogenetic tests demonstrated that salmonid diversification patterns are closely allied in time with the continuous climatic cooling that followed the Eocene–Oligocene transition, with the highest diversification rates coinciding with recent ice ages. Further tests revealed considerably higher speciation rates in lineages that evolved anadromy—the physiological capacity to migrate between fresh and seawater—than in sister groups that retained the ancestral state of freshwater residency. Anadromy, which probably evolved in response to climatic cooling, is an established catalyst of genetic isolation, particularly during environmental perturbations (for example, glaciation cycles). We thus conclude that climate-linked ecophysiological factors, rather than WGD, were the primary drivers of salmonid diversification. PMID:24452024

  11. Impaired integrity of the brain parenchyma in non-geriatric patients with major depressive disorder revealed by diffusion tensor imaging.

    PubMed

    Tha, Khin K; Terae, Satoshi; Nakagawa, Shin; Inoue, Takeshi; Kitagawa, Nobuki; Kako, Yuki; Nakato, Yasuya; Akter Popy, Kawser; Fujima, Noriyuki; Zaitsu, Yuri; Yoshida, Daisuke; Ito, Yoichi M; Miyamoto, Tamaki; Koyama, Tsukasa; Shirato, Hiroki

    2013-06-30

    Diffusion tensor imaging (DTI) is considered to be able to non-invasively quantify white matter integrity. This study aimed to use DTI to evaluate white matter integrity in non-geriatric patients with major depressive disorder (MDD) who were free of antidepressant medication. DTI was performed on 19 non-geriatric patients with MDD, free of antidepressant medication, and 19 age-matched healthy subjects. Voxel-based and histogram analyses were used to compare fractional anisotropy (FA) and mean diffusivity (MD) values between the two groups, using two-sample t tests. The abnormal DTI indices, if any, were tested for correlation with disease duration and severity, using Pearson product-moment correlation analysis. Voxel-based analysis showed clusters with FA decrease at the bilateral frontal white matter, anterior limbs of internal capsule, cerebellum, left putamen and right thalamus of the patients. Histogram analysis revealed lower peak position of FA histograms in the patients. FA values of the abnormal clusters and peak positions of FA histograms of the patients exhibited moderate correlation with disease duration and severity. These results suggest the implication of frontal-subcortical circuits and cerebellum in MDD, and the potential utility of FA in evaluation of brain parenchymal integrity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  12. Molecular phenotyping of immune cells from young NOD mice reveals abnormal metabolic pathways in the early induction phase of autoimmune diabetes.

    PubMed

    Wu, Jian; Kakoola, Dorothy N; Lenchik, Nataliya I; Desiderio, Dominic M; Marshall, Dana R; Gerling, Ivan C

    2012-01-01

    Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse--a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (~90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes.

  13. GENE EXPRESSION PROFILING IN AGING RATS AND MICE REVEALS CHANGES IN XENOBIOTIC METABOLISM GENES

    EPA Science Inventory

    Detoxification and elimination of xenobiotics are major functions of the liver and is important in maintaining the metabolic homeostasis of the organism. The degree to which aging affects hepatic metabolism is not known. The expression of xenobiotic metabolizing enzymes (XMEs), i...

  14. Coadminstration of L. major amastigote class I nuclease (rLmaCIN) with LPD nanoparticles delays the progression of skin lesion and the L. major dissemination to the spleen in BALB/c mice-based experimental setting.

    PubMed

    Fakhraee, Fatemeh; Badiee, Ali; Alavizadeh, Seyedeh Hoda; Jalali, Seyed Amir; Chavoshian, Omid; Khamesipour, Ali; Mahboudi, Fereidoun; Jaafari, Mahmoud Reza

    2016-07-01

    Human cutaneous leishmaniasis is a disease caused by eukaryotic single-celled Leishmania species, the developmental program of which relies upon blood-feeding adult female sand flies and their dominant mammal blood sources, namely wild rodents in area where human beings exert more or less transient activities. The recourse to model rodents - namely laboratory mice such as C57BL/6 mice - has allowed extracted the immune signatures that account for the healing of the transient cutaneous lesion that develops at the site where Leishmania major promastigotes were delivered. Indeed, if the latter mice are exposed to a second inoculum of L. major promastigotes, no lesion will develop in the secondary skin site remodeled as a niche for a low size intracellular L. major amastigote population. Moreover, IFN-γ dominates over IL-10 in the supernatant of cultures of PBMCs -prepared from blood sampled from human beings who healed from a cutaneous lesion- and incubated with L. major class I Nuclease LmaCIN, a protein highly expressed in the cell-cycling amastigote population which is dominant by macrophages. Altogether, these datasets were strong incentive to promote research aimed to design and monitor efficacy of L. major amastigote protein-based vaccines in pre-clinical settings. Using L. major enzyme class I nuclease (LmaCIN) expressed in the L. major cell-cycling amastigote population hosted by macrophages, BALB/c mice were immunized three times with either rLmaCIN plus LPD nanoparticles (LPD-rLmaCIN), or rLmaCIN-CpG DNA or free rLmaCIN and dextrose. The following parameters: footpad swelling, splenic L. major load, L. major binding IgGs and cytokine profiles of rLmaCIN- reactive T lymphocytes were then compared. Once coadminstered with LPD, rLmaCIN allow BALB/c mice to display delayed onset of skin lesion at the challenge inoculation site and delayed L. major dissemination from the challenged site to the spleen. Thus, the LPD-rLmaCIN is shown to display some promising

  15. Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice.

    PubMed

    Gong, Ping; Roseman, Jelita; Fernandez, Celia G; Vetrivel, Kulandaivelu S; Bindokas, Vytautas P; Zitzow, Lois A; Kar, Satyabrata; Parent, Angèle T; Thinakaran, Gopal

    2011-12-28

    p23 belongs to the highly conserved p24 family of type I transmembrane proteins, which participate in the bidirectional protein transport between the endoplasmic reticulum and Golgi apparatus. Mammalian p23 has been shown to interact with γ-secretase complex, and modulate secretory trafficking as well as intramembranous processing of amyloid precursor protein in cultured cells. Negative modulation of β-amyloid production by p23 in cultured cell lines suggested that elevation of p23 expression in neurons might mitigate cerebral amyloid burden. We generated several lines of transgenic mice expressing human p23 in neurons under the control of Thy-1.2 promoter. We found that even a 50% increase in p23 levels in the central nervous system of mice causes post-natal growth retardation, severe neurological problems characterized by tremors, seizure, ataxia, and uncoordinated movements, and premature death. The severity of the phenotype closely correlated with the level of p23 overexpression in multiple transgenic lines. While the number and general morphology of neurons in Hup23 mice appeared to be normal throughout the brain, abnormal non-Golgi p23 localization was observed in a subset of neurons with high transgene expression in brainstem. Moreover, detailed immunofluorescence analysis revealed marked proliferation of astrocytes, activation of microglia, and thinning of myelinated bundles in brainstem of Hup23 mice. These results demonstrate that proper level of p23 expression is critical for neuronal function, and perturbing p23 function by overexpression initiates a cascade of cellular reactions in brainstem that leads to severe motor deficits and other neurological problems, which culminate in premature death. The neurological phenotype observed in Hup23 mice highlights significant adverse effects associated with manipulating neuronal expression of p23, a previously described negative modulator of γ-secretase activity and β-amyloid production. Moreover, our report

  16. Arid1b haploinsufficient mice reveal neuropsychiatric phenotypes and reversible causes of growth impairment

    PubMed Central

    Celen, Cemre; Chuang, Jen-Chieh; Luo, Xin; Nijem, Nadine; Walker, Angela K; Chen, Fei; Zhang, Shuyuan; Chung, Andrew S; Nguyen, Liem H; Nassour, Ibrahim; Budhipramono, Albert; Sun, Xuxu; Bok, Levinus A; McEntagart, Meriel; Gevers, Evelien F; Birnbaum, Shari G; Eisch, Amelia J; Powell, Craig M; Ge, Woo-Ping; Santen, Gijs WE; Chahrour, Maria; Zhu, Hao

    2017-01-01

    Sequencing studies have implicated haploinsufficiency of ARID1B, a SWI/SNF chromatin-remodeling subunit, in short stature (Yu et al., 2015), autism spectrum disorder (O'Roak et al., 2012), intellectual disability (Deciphering Developmental Disorders Study, 2015), and corpus callosum agenesis (Halgren et al., 2012). In addition, ARID1B is the most common cause of Coffin-Siris syndrome, a developmental delay syndrome characterized by some of the above abnormalities (Santen et al., 2012; Tsurusaki et al., 2012; Wieczorek et al., 2013). We generated Arid1b heterozygous mice, which showed social behavior impairment, altered vocalization, anxiety-like behavior, neuroanatomical abnormalities, and growth impairment. In the brain, Arid1b haploinsufficiency resulted in changes in the expression of SWI/SNF-regulated genes implicated in neuropsychiatric disorders. A focus on reversible mechanisms identified Insulin-like growth factor (IGF1) deficiency with inadequate compensation by Growth hormone-releasing hormone (GHRH) and Growth hormone (GH), underappreciated findings in ARID1B patients. Therapeutically, GH supplementation was able to correct growth retardation and muscle weakness. This model functionally validates the involvement of ARID1B in human disorders, and allows mechanistic dissection of neurodevelopmental diseases linked to chromatin-remodeling. DOI: http://dx.doi.org/10.7554/eLife.25730.001 PMID:28695822

  17. Modulating dopamine release by optogenetics in transgenic mice reveals terminal dopaminergic dynamics

    PubMed Central

    Lu, Yao; Driscoll, Nicolette; Ozden, Ilker; Yu, Zeyang; Nurmikko, Arto V.

    2015-01-01

    Abstract. Dopamine (DA) release and uptake dynamics in the nucleus accumbens (NAc) have important implications for neurological diseases and mammalian animal behaviors. We demonstrate here the use of cell-type-specific optogenetic targeting in conjunction with fast-scan cyclic voltammetry applied to brain slices prepared from specifically tailored transgenic mice, which conditionally express channelrhodopsin-2 (ChR2) through dopamine transporter (DAT)-Cre. Terminal dopaminergic dynamics and the direct manipulation of induced DA release level by controlling light intensity, pulse width, and the shape of stimulation waveforms were studied. Effective cell terminal-targeting optogenetic induction of DA release at physiological levels in NAc is demonstrated and discussed. It was found that delivering more light energy by increasing stimulation intensity and length is not the only way to control DA release; the temporal shape of the stimulus waveform at light onset is also critically related to induced DA concentrations. In addition, DA uptake dynamics as well as the recovery of the presynaptic releasable DA pool are studied and modeled. More broadly, our experimental findings provide important further evidence for effectively applying optogenetics to induce neurotransmitter release in the behaviorally relevant region of the brain in a highly cell-type selective context. PMID:26171413

  18. Stereocilin-deficient mice reveal the origin of cochlear waveform distortions.

    PubMed

    Verpy, Elisabeth; Weil, Dominique; Leibovici, Michel; Goodyear, Richard J; Hamard, Ghislaine; Houdon, Carine; Lefèvre, Gaelle M; Hardelin, Jean-Pierre; Richardson, Guy P; Avan, Paul; Petit, Christine

    2008-11-13

    Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.

  19. Analysis of morphine responses in mice reveals a QTL on Chromosome 7

    PubMed Central

    Crusio, Wim E.; Dhawan, Esha; Chesler, Elissa J.; Delprato, Anna

    2016-01-01

    In this study we identified a quantitative trait locus (QTL) on mouse Chromosome 7 associated with locomotor activity and rearing post morphine treatment. This QTL was revealed after correcting for the effects of another QTL peak on Chromosome 10 using composite interval mapping. The positional candidate genes are Syt9 and Ppfibp2. Several other genes within the interval are linked to neural processes, locomotor activity, and the defensive response to harmful stimuli. PMID:27746909

  20. Analysis of morphine responses in mice reveals a QTL on Chromosome 7.

    PubMed

    Crusio, Wim E; Dhawan, Esha; Chesler, Elissa J; Delprato, Anna

    2016-01-01

    In this study we identified a quantitative trait locus (QTL) on mouse Chromosome 7 associated with locomotor activity and rearing post morphine treatment. This QTL was revealed after correcting for the effects of another QTL peak on Chromosome 10 using composite interval mapping. The positional candidate genes are Syt9 and Ppfibp2. Several other genes within the interval are linked to neural processes, locomotor activity, and the defensive response to harmful stimuli.

  1. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    SciTech Connect

    Shi, CY; Yang, H; Wei, CL; Yu, O; Zhang, ZZ; Sun, J; Wan, XC

    2011-01-01

    Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real

  2. Expression Profiling Reveals Novel Hypoxic Biomarkers in Peripheral Blood of Adult Mice Exposed to Chronic Hypoxia

    PubMed Central

    Zeiger, Ulrike; Khurana, Tejvir S.

    2012-01-01

    Hypoxia induces a myriad of changes including an increase in hematocrit due to erythropoietin (EPO) mediated erythropoiesis. While hypoxia is of importance physiologically and clinically, lacunae exist in our knowledge of the systemic and temporal changes in gene expression occurring in blood during the exposure and recovery from hypoxia. To identify these changes expression profiling was conducted on blood obtained from cohorts of C57Bl-10 wild type mice that were maintained at normoxia (NX), exposed for two weeks to normobaric chronic hypoxia (CH) or two weeks of CH followed by two weeks of normoxic recovery (REC). Using stringent bioinformatic cut-offs (0% FDR, 2 fold change cut-off), 230 genes were identified and separated into four distinct temporal categories. Class I) contained 1 transcript up-regulated in both CH and REC; Class II) contained 202 transcripts up-regulated in CH but down-regulated after REC; Class III) contained 9 transcripts down-regulated both in CH and REC; Class IV) contained 18 transcripts down-regulated after CH exposure but up-regulated after REC. Profiling was independently validated and extended by analyzing expression levels of selected genes as novel biomarkers from our profile (e.g. spectrin alpha-1, ubiquitin domain family-1 and pyrroline-5-carboxylate reductase-1) by performing qPCR at 7 different time points during CH and REC. Our identification and characterization of these genes define transcriptome level changes occurring during chronic hypoxia and normoxic recovery as well as novel blood biomarkers that may be useful in monitoring a variety of physiological and pathological conditions associated with hypoxia. PMID:22629407

  3. Hair Regenerative Mechanisms of Red Ginseng Oil and Its Major Components in the Testosterone-Induced Delay of Anagen Entry in C57BL/6 Mice.

    PubMed

    Truong, Van-Long; Bak, Min Ji; Lee, Changook; Jun, Mira; Jeong, Woo-Sik

    2017-09-08

    Hair loss (alopecia) is a universal problem for numerous people in the world. The present study was conducted to investigate the effects of red ginseng oil (RGO) and its major components on hair re-growth using testosterone (TES)-induced delay of anagen entry in C57BL/6 mice and their mechanisms of action. Seven-week-old C57BL/6 mice were daily treated with TES for 1 h prior to topical application of 10% RGO, 1% linoleic acid (LA), 1% β-sitosterol (SITOS), or 1% bicyclo(10.1.0)tridec-1-ene (BICYCLO) once a day for 28 days. Hair regenerative capacity was significantly restored by treatment of RGO and its major compounds in the TES-treated mice. Histological analysis showed that RGO along with LA and SITOS but not BICYCLO promoted hair growth through early inducing anagen phase that was delayed by TES in mice. Treatment of mice with RGO, LA, or SITOS up-regulated Wnt/β-catenin and Shh/Gli pathways-mediated expression of genes such as β-catenin, Lef-1, Sonic hedgehog, Smoothened, Gli-1, Cyclin D1, and Cyclin E in the TES-treated mice. In addition, RGO and its major components reduced the protein level of TGF-β but enhanced the expression of anti-apoptotic protein Bcl-2. These results suggest that RGO is a potent novel therapeutic natural product for treatment of androgenic alopecia possibly through hair re-growth activity of its major components such as LA and SITOS.

  4. Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function.

    PubMed

    Zhang, J C; Kim, S; Helmke, B P; Yu, W W; Du, K L; Lu, M M; Strobeck, M; Yu, Q; Parmacek, M S

    2001-02-01

    SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

  5. Applying the ethoexperimental approach to neurodevelopmental syndrome research reveals exaggerated defensive behavior in Mecp2 mutant mice.

    PubMed

    Pearson, Brandon L; Defensor, Erwin B; Blanchard, D Caroline; Blanchard, Robert J

    2015-07-01

    Rett syndrome is a Pervasive Developmental Disorder (PDD) associated with de novo mutations of the methyl CpG-binding protein 2 (MECP2) gene. Mecp2 functions as a transcription factor that regulates the expression of hundreds of genes. Identification of the role of Mecp2 in specific neurodevelopmental symptoms remains an important research aim. We previously demonstrated that male mice possessing a truncation mutation in Mecp2 are hyper-social. We predicted that reduced fear or anxiety might underlie this enhanced affiliation. In order to probe risk assessment and anxiety-like behavior, we compared Mecp2 truncation mutants to their wild-type littermates in the elevated plus maze and elevated zero maze. Additionally, subjects were administered the mouse defense test battery to evaluate unconditioned fear- and panic-like behavior to a graded set of threat scenarios and a predator stimulus. Mutant mice showed no significant changes in anxiety-like behavior. Yet, they displayed hyper-reactive escape and defensive behaviors to an animate predatory threat stimulus. Notably, mutant mice engaged in exaggerated active defense responding to threat stimuli at nearly all phases of the fear battery. These results reveal abnormalities in emotion regulation in Mecp2 mutants particularly in response to ecologically relevant threats. This hyper-responsivity suggests that transcriptional targets of Mecp2 are critical to emotion regulation. Moreover, we suggest that detailed analysis of defensive behavior and aggression with ethologically relevant tasks provides an avenue to interrogate gene-behavior mechanisms of neurodevelopmental and other psychiatric conditions.

  6. Analysis of Uncoupling Protein 2-Deficient Mice upon Anaesthesia and Sedation Revealed a Role for UCP2 in Locomotion

    PubMed Central

    Alves-Guerra, Marie-Clotilde; Aheng, Caroline; Pecqueur, Claire; Masscheleyn, Sandrine; Tharaux, Pierre Louis; Druilhe, Anne; Ricquier, Daniel; Challet, Etienne; Miroux, Bruno

    2012-01-01

    General anaesthesia is associated with hypothermia, oxidative stress, and immune depression. Uncoupling Protein (UCP2) is a member of the mitochondrial carrier family present in many organs including the spleen, the lung and the brain. A role of UCP2 in the activation of the inflammatory/immune cells, in the secretion of hormones, and in the excitability of neurons by regulating the production of reactive oxygen species has been discussed. Because of the side effects of anaesthesia listed above, we aimed to question the expression and the function of UCP2 during anaesthesia. Induction of anaesthesia with ketamine (20 mg/kg) or isoflurane (3.6%) and induction of sedation with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg) stimulated infiltration of immune cells in the lung and increased UCP2 protein content in the lung, in both immune and non-immune cells. UCP2 content in the lung inversely correlated with body temperature decrease induced by medetomidine treatment. Challenge of the Ucp2−/− mice with isoflurane and medetomidine revealed an earlier behavioral recovery phenotype. Transponder analysis of body temperature and activity showed no difference between Ucp2−/− and control mice in basal conditions. However, upon an acute decrease of body temperature induced by medetomidine, Ucp2−/− mice exhibited increased locomotion activity. Together, these results show that UCP2 is rapidly mobilized during anaesthesia and sedation in immune cells, and suggest a role of UCP2 in locomotion. PMID:22900002

  7. Pathway Network Analyses for Autism Reveal Multisystem Involvement, Major Overlaps with Other Diseases and Convergence upon MAPK and Calcium Signaling

    PubMed Central

    Wen, Ya; Alshikho, Mohamad J.; Herbert, Martha R.

    2016-01-01

    We used established databases in standard ways to systematically characterize gene ontologies, pathways and functional linkages in the large set of genes now associated with autism spectrum disorders (ASDs). These conditions are particularly challenging—they lack clear pathognomonic biological markers, they involve great heterogeneity across multiple levels (genes, systemic biological and brain characteristics, and nuances of behavioral manifestations)—and yet everyone with this diagnosis meets the same defining behavioral criteria. Using the human gene list from Simons Foundation Autism Research Initiative (SFARI) we performed gene set enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database, and then derived a pathway network from pathway-pathway functional interactions again in reference to KEGG. Through identifying the GO (Gene Ontology) groups in which SFARI genes were enriched, mapping the coherence between pathways and GO groups, and ranking the relative strengths of representation of pathway network components, we 1) identified 10 disease-associated and 30 function-associated pathways 2) revealed calcium signaling pathway and neuroactive ligand-receptor interaction as the most enriched, statistically significant pathways from the enrichment analysis, 3) showed calcium signaling pathways and MAPK signaling pathway to be interactive hubs with other pathways and also to be involved with pervasively present biological processes, 4) found convergent indications that the process “calcium-PRC (protein kinase C)-Ras-Raf-MAPK/ERK” is likely a major contributor to ASD pathophysiology, and 5) noted that perturbations associated with KEGG’s category of environmental information processing were common. These findings support the idea that ASD-associated genes may contribute not only to core features of ASD themselves but also to vulnerability to other chronic and systemic problems potentially including cancer, metabolic

  8. Proteome Analysis of Streptococcus thermophilus Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein

    PubMed Central

    Derzelle, Sylviane; Bolotin, Alexander; Mistou, Michel-Yves; Rul, Françoise

    2005-01-01

    We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes. PMID:16332852

  9. Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous–Paleogene boundary

    PubMed Central

    Feng, Yan-Jie; Liang, Dan; Hillis, David M.; Cannatella, David C.; Zhang, Peng

    2017-01-01

    Frogs (Anura) are one of the most diverse groups of vertebrates and comprise nearly 90% of living amphibian species. Their worldwide distribution and diverse biology make them well-suited for assessing fundamental questions in evolution, ecology, and conservation. However, despite their scientific importance, the evolutionary history and tempo of frog diversification remain poorly understood. By using a molecular dataset of unprecedented size, including 88-kb characters from 95 nuclear genes of 156 frog species, in conjunction with 20 fossil-based calibrations, our analyses result in the most strongly supported phylogeny of all major frog lineages and provide a timescale of frog evolution that suggests much younger divergence times than suggested by earlier studies. Unexpectedly, our divergence-time analyses show that three species-rich clades (Hyloidea, Microhylidae, and Natatanura), which together comprise ∼88% of extant anuran species, simultaneously underwent rapid diversification at the Cretaceous–Paleogene (K–Pg) boundary (KPB). Moreover, anuran families and subfamilies containing arboreal species originated near or after the KPB. These results suggest that the K–Pg mass extinction may have triggered explosive radiations of frogs by creating new ecological opportunities. This phylogeny also reveals relationships such as Microhylidae being sister to all other ranoid frogs and African continental lineages of Natatanura forming a clade that is sister to a clade of Eurasian, Indian, Melanesian, and Malagasy lineages. Biogeographical analyses suggest that the ancestral area of modern frogs was Africa, and their current distribution is largely associated with the breakup of Pangaea and subsequent Gondwanan fragmentation. PMID:28673970

  10. Gene Expression Analysis of Rice Seedling under Potassium Deprivation Reveals Major Changes in Metabolism and Signaling Components

    PubMed Central

    Shankar, Alka; Singh, Amarjeet; Kanwar, Poonam; Srivastava, Ashish Kumar; Pandey, Amita; Suprasanna, Penna; Kapoor, Sanjay; Pandey, Girdhar K.

    2013-01-01

    Plant nutrition is one of the important areas for improving the yield and quality in crops as well as non-crop plants. Potassium is an essential plant nutrient and is required in abundance for their proper growth and development. Potassium deficiency directly affects the plant growth and hence crop yield and production. Recently, potassium-dependent transcriptomic analysis has been performed in the model plant Arabidopsis, however in cereals and crop plants; such a transcriptome analysis has not been undertaken till date. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency then replenished with potassium. Our results reveal that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K+. Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants. PMID:23922980

  11. Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice

    PubMed Central

    Popova, Taissia G.; Espina, Virginia; Liotta, Lance A.; Popov, Serguei G.

    2015-01-01

    Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissesction. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and

  12. Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

    PubMed

    Popova, Taissia G; Espina, Virginia; Liotta, Lance A; Popov, Serguei G

    2015-01-01

    Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and

  13. Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice

    PubMed Central

    Hartmann, David A.; Underly, Robert G.; Grant, Roger I.; Watson, Ashley N.; Lindner, Volkhard; Shih, Andy Y.

    2015-01-01

    Abstract. Pericytes are essential for normal brain function, but many aspects of their physiology remain enigmatic due to a lack of tools to genetically target this cell population. Here, we characterize brain pericytes using two existing Cre-recombinase driver mouse lines that can serve distinct purposes in cerebrovascular research. One line expresses an inducible version of Cre under the NG2 proteoglycan promoter, which provides the sparse labeling necessary to define the morphology of single cells. These mice reveal structural differences between pericytes adjacent to arterioles versus those broadly distributed in the capillary bed that may underlie differential roles in control of vessel caliber. A second line expresses Cre constitutively under the platelet-derived growth factor receptor β promoter and provides continuous, highly specific and near-complete labeling of pericytes and myocytes along the entire cerebrovasculature. This line provides a three-dimensional view of pericyte distribution along the cortical angioarchitecture following optical clearing of brain tissue. In combination with recent reporter lines for expression of optogenetic actuators and activity-sensitive probes, these mice may be key tools for studying pericyte biology in the intact brain. PMID:26158016

  14. Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

    PubMed Central

    Bultmann-Mellin, Insa; Conradi, Anne; Maul, Alexandra C.; Dinger, Katharina; Wempe, Frank; Wohl, Alexander P.; Imhof, Thomas; Wunderlich, F. Thomas; Bunck, Alexander C.; Nakamura, Tomoyuki; Koli, Katri; Bloch, Wilhelm; Ghanem, Alexander; Heinz, Andrea; von Melchner, Harald; Sengle, Gerhard; Sterner-Kock, Anja

    2015-01-01

    Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/−) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C. PMID:25713297

  15. The glycerophospho-metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice

    PubMed Central

    Kopp, Florian; Komatsu, Toru; Nomura, Daniel K.; Trauger, Sunia A.; Thomas, Jason R.; Siuzdak, Gary; Simon, Gabriel M.; Cravatt, Benjamin F.

    2010-01-01

    GDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophopho-inositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (> 20-fold) elevated in brain tissue from GDE1(-/-) mice. Furthermore, two additional GroP-metabolites not previously identified in eukaryotic cells, glycerophospho-serine (GroPSer) and glycerophospho-glycerate (GroPGate), were also highly elevated in GDE1(-/-) brains. Enzyme assays with synthetic GroP-metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1 (-/-) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo. PMID:20797612

  16. Generation of a microglial developmental index in mice and in humans reveals a sex difference in maturation and immune reactivity.

    PubMed

    Hanamsagar, Richa; Alter, Mark D; Block, Carina S; Sullivan, Haley; Bolton, Jessica L; Bilbo, Staci D

    2017-09-01

    Evidence suggests many neurological disorders emerge when normal neurodevelopmental trajectories are disrupted, i.e., when circuits or cells do not reach their fully mature state. Microglia play a critical role in normal neurodevelopment and are hypothesized to contribute to brain disease. We used whole transcriptome profiling with Next Generation sequencing of purified developing microglia to identify a microglial developmental gene expression program involving thousands of genes whose expression levels change monotonically (up or down) across development. Importantly, the gene expression program was delayed in males relative to females and exposure of adult male mice to LPS, a potent immune activator, accelerated microglial development in males. Next, a microglial developmental index (MDI) generated from gene expression patterns obtained from purified mouse microglia, was applied to human brain transcriptome datasets to test the hypothesis that variability in microglial development is associated with human diseases such as Alzheimer's and autism where microglia have been suggested to play a role. MDI was significantly increased in both Alzheimer's Disease and in autism, suggesting that accelerated microglial development may contribute to neuropathology. In conclusion, we identified a microglia-specific gene expression program in mice that was used to create a microglia developmental index, which was applied to human datasets containing heterogeneous cell types to reveal differences between healthy and diseased brain samples, and between males and females. This powerful tool has wide ranging applicability to examine microglial development within the context of disease and in response to other variables such as stress and pharmacological treatments. © 2017 Wiley Periodicals, Inc.

  17. Synaptic Changes in the Dentate Gyrus of APP/PS1 Transgenic Mice Revealed by Electron Microscopy

    PubMed Central

    Merino-Serrais, Paula; Gonzalez, Santiago; DeFelipe, Javier

    2013-01-01

    Abstract Numerous studies have reported widespread synaptic dysfunction or loss in early stages of both Alzheimer disease (AD) patients and animal models; it is widely accepted that synapse loss is the major structural correlate of cognitive dysfunction. Elucidation of the changes that may affect synapses is crucial for understanding the pathogenic mechanisms underlying AD, but ultrastructural preservation of human postmortem brain tissue is often poor, and classical methods for quantification of synapses have significant technical limitations. We previously observed changes in dendritic spines in plaque-free regions of the neuropil of the dentate gyrus of double-transgenic APP/PS1 (amyloid precursor protein/presenilin 1) model mice by light microscopy. Here, we used electron microscopy to examine possible synaptic alterations in this region. We used standard stereologic techniques to determine numbers of synapses per volume. We were able to reconstruct and analyze thousands of synapses and their 3-dimensional characteristics using a focused ion beam/scanning electron microscope and 3-dimensional reconstruction software (EspINA), which performs semiautomated segmentation of synapses. Our results show that both numbers of synapses per volume and synaptic morphology are affected in plaque-free regions of APP/PS1 mice. Therefore, changes in the number and morphology of synapses seem to be widespread alterations in this animal model. PMID:23584198

  18. Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination

    PubMed Central

    Ehrenstein, Michael R.; Rada, Cristina; Jones, Anne-Marie; Milstein, César; Neuberger, Michael S.

    2001-01-01

    Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution. PMID:11717399

  19. Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

    PubMed Central

    White, Jacqueline K.; Gerdin, Anna-Karin; Karp, Natasha A.; Ryder, Ed; Buljan, Marija; Bussell, James N.; Salisbury, Jennifer; Clare, Simon; Ingham, Neil J.; Podrini, Christine; Houghton, Richard; Estabel, Jeanne; Bottomley, Joanna R.; Melvin, David G.; Sunter, David; Adams, Niels C.; Baker, Lauren; Barnes, Caroline; Beveridge, Ryan; Cambridge, Emma; Carragher, Damian; Chana, Prabhjoat; Clarke, Kay; Hooks, Yvette; Igosheva, Natalia; Ismail, Ozama; Jackson, Hannah; Kane, Leanne; Lacey, Rosalind; Lafont, David Tino; Lucas, Mark; Maguire, Simon; McGill, Katherine; McIntyre, Rebecca E.; Messager, Sophie; Mottram, Lynda; Mulderrig, Lee; Pearson, Selina; Protheroe, Hayley J.; Roberson, Laura-Anne; Salsbury, Grace; Sanderson, Mark; Sanger, Daniel; Shannon, Carl; Thompson, Paul C.; Tuck, Elizabeth; Vancollie, Valerie E.; Brackenbury, Lisa; Bushell, Wendy; Cook, Ross; Dalvi, Priya; Gleeson, Diane; Habib, Bishoy; Hardy, Matt; Liakath-Ali, Kifayathullah; Miklejewska, Evelina; Price, Stacey; Sethi, Debarati; Trenchard, Elizabeth; von Schiller, Dominique; Vyas, Sapna; West, Anthony P.; Woodward, John; Wynn, Elizabeth; Evans, Arthur; Gannon, David; Griffiths, Mark; Holroyd, Simon; Iyer, Vivek; Kipp, Christian; Lewis, Morag; Li, Wei; Oakley, Darren; Richardson, David; Smedley, Damian; Agu, Chukwuma; Bryant, Jackie; Delaney, Liz; Gueorguieva, Nadia I.; Tharagonnet, Helen; Townsend, Anne J.; Biggs, Daniel; Brown, Ellen; Collinson, Adam; Dumeau, Charles-Etienne; Grau, Evelyn; Harrison, Sarah; Harrison, James; Ingle, Catherine E.; Kundi, Helen; Madich, Alla; Mayhew, Danielle; Metcalf, Tom; Newman, Stuart; Pass, Johanna; Pearson, Laila; Reynolds, Helen; Sinclair, Caroline; Wardle-Jones, Hannah; Woods, Michael; Alexander, Liam; Brown, Terry; Flack, Francesca; Frost, Carole; Griggs, Nicola; Hrnciarova, Silvia; Kirton, Andrea; McDermott, Jordan; Rogerson, Claire; White, Gemma; Zielezinski, Pawel; DiTommaso, Tia; Edwards, Andrew; Heath, Emma; Mahajan, Mary Ann; Yalcin, Binnaz; Tannahill, David; Logan, Darren W.; MacArthur, Daniel G.; Flint, Jonathan; Mahajan, Vinit B.; Tsang, Stephen H.; Smyth, Ian; Watt, Fiona M.; Skarnes, William C.; Dougan, Gordon; Adams, David J.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.

    2013-01-01

    Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip PMID:23870131

  20. Spontaneous voiding by mice reveals strain-specific lower urinary tract function to be a quantitative genetic trait

    PubMed Central

    Yu, Weiqun; Ackert-Bicknell, Cheryl; Larigakis, John D.; MacIver, Bryce; Steers, William D.; Churchill, Gary A.; Hill, Warren G.

    2014-01-01

    Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18–24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations. PMID:24717733

  1. Hippo signaling pathway reveals a spatio-temporal correlation with the size of primordial follicle pool in mice.

    PubMed

    Xiang, Cheng; Li, Jia; Hu, Liaoliao; Huang, Jian; Luo, Tao; Zhong, Zhisheng; Zheng, Yuehui; Zheng, Liping

    2015-01-01

    The Hippo signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms and regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Hippo signaling are expressed in mammalian ovaries, but the exact functions of this pathway in primordial follicle development remains unclear. To analyze the spatio-temporal correlation between the core component of Hippo pathway and the size of primordial follicle pool, Western blot, Real-time PCR and immunohistochemistry were used, and the expression and localization of MST1, LATS2 and YAP1 mRNA and protein were examined in 3 d, 1 m, 5 m, 16 m postnatal mice ovary and the culture model of mice primordial follicle in vitro. Both the protein and mRNA expression of the MST1 and LATS2 were decreased significantly as mouse age increased (p < 0.05), however, the mRNA expression of them increased significantly in 16 m compared with 5 m as well as the protein expression of LATS2.The expression of YAP showed the opposite trend, and the significant protein expression of pYAP was increased before 1 m, after which no significant change was observed. Moreover, the ratio of pYAP/YAP decreased significantly. Culturing ovaries for 8 d in vitro resulted in the activation of primordial follicles in 3 d postnatal mice ovaries, and these developed into primary follicles with the expression of PCNA increasing significantly (p < 0.05). The mRNA and protein expression of MST and LATS decreased significantly (p < 0.05), and the expression of YAP increased significantly (p < 0.05, p < 0.01), whereas the ratio of pYAP/YAP decreased significantly (p < 0.05). The above results reveal that the expression of the core components of Hippo pathway changed during mouse follicular development, especially before and after primordial follicle activation in vitro. The primordial follicle activation may be related to the significant decrease of the ratio of pYAP1/YAP1. In conclusion

  2. Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions

    PubMed Central

    2012-01-01

    Background The biphasic life cycle with pelagic larva and benthic adult stages is widely observed in the animal kingdom, including the Porifera (sponges), which are the earliest branching metazoans. The demosponge, Amphimedon queenslandica, undergoes metamorphosis from a free-swimming larva into a sessile adult that bears no morphological resemblance to other animals. While the genome of A. queenslandica contains an extensive repertoire of genes very similar to that of complex bilaterians, it is as yet unclear how this is drawn upon to coordinate changing morphological features and ecological demands throughout the sponge life cycle. Results To identify genome-wide events that accompany the pelagobenthic transition in A. queenslandica, we compared global gene expression profiles at four key developmental stages by sequencing the poly(A) transcriptome using SOLiD technology. Large-scale changes in transcription were observed as sponge larvae settled on the benthos and began metamorphosis. Although previous systematics suggest that the only clear homology between Porifera and other animals is in the embryonic and larval stages, we observed extensive use of genes involved in metazoan-associated cellular processes throughout the sponge life cycle. Sponge-specific transcripts are not over-represented in the morphologically distinct adult; rather, many genes that encode typical metazoan features, such as cell adhesion and immunity, are upregulated. Our analysis further revealed gene families with candidate roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules. Conclusions This first genome-wide study of the developmental transcriptome in an early branching metazoan highlights major transcriptional events that accompany the pelagobenthic transition and point to a network of regulatory mechanisms that coordinate changes in morphology with shifting environmental demands

  3. Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

    PubMed

    White, Jacqueline K; Gerdin, Anna-Karin; Karp, Natasha A; Ryder, Ed; Buljan, Marija; Bussell, James N; Salisbury, Jennifer; Clare, Simon; Ingham, Neil J; Podrini, Christine; Houghton, Richard; Estabel, Jeanne; Bottomley, Joanna R; Melvin, David G; Sunter, David; Adams, Niels C; Tannahill, David; Logan, Darren W; Macarthur, Daniel G; Flint, Jonathan; Mahajan, Vinit B; Tsang, Stephen H; Smyth, Ian; Watt, Fiona M; Skarnes, William C; Dougan, Gordon; Adams, David J; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P

    2013-07-18

    Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP: Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Controlled DNA double-strand break induction in mice reveals post-damage transcriptome stability.

    PubMed

    Kim, Jeongkyu; Sturgill, David; Tran, Andy D; Sinclair, David A; Oberdoerffer, Philipp

    2016-04-20

    DNA double-strand breaks (DSBs) and their repair can cause extensive epigenetic changes. As a result, DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways. Studying the consequences of DSBs in higher organisms has, however, been hindered by a scarcity of tools for controlled DSB induction. Here, we describe a mouse model that allows for both tissue-specific and temporally controlled DSB formation at ∼140 defined genomic loci. Using this model, we show that DSBs promote a DNA damage signaling-dependent decrease in gene expression in primary cells specifically at break-bearing genes, which is reversed upon DSB repair. Importantly, we demonstrate that restoration of gene expression can occur independently of cell cycle progression, underlining its relevance for normal tissue maintenance. Consistent with this, we observe no evidence for persistent transcriptional repression in response to a multi-day course of continuous DSB formation and repair in mouse lymphocytes in vivo Together, our findings reveal an unexpected capacity of primary cells to maintain transcriptome integrity in response to DSBs, pointing to a limited role for DNA damage as a mediator of cell-autonomous epigenetic dysfunction.

  5. Effect of trinitroglycerin therapy on serum zinc and copper levels and liver enzyme activities in BALB/c mice infected with Leishmania major MRHO/IR/75/ER

    PubMed Central

    Najafzade, Mana; Mosapour, Abbas; Nahrevanian, Hossein; Zamani, Zahra; Javadian, Seifoddin; Mirkhani, Fatemeh

    2015-01-01

    Objective(s): To evaluate the effect of trinitroglycerin (TNG) as nitric oxide donor agent on serum copper (Cu) and zinc (Zn) levels and liver enzymes in BALB/c mice infected with Leishmania major (L. major) MRHO/IR/75/ER. Materials and Methods: Inbred female mice were divided into three groups: healthy group (uninfected naive mice), control group (infected with L. major), and test group (L. major infected mice treated with TNG). TNG (200 µg/µl) was inoculated subcutaneously into the mice of the test group. Serum Cu and Zn levels and liver enzymes activities were then evaluated by atomic absorption spectrophometer and colorimetric methods, respectively. Results: Serum Cu levels were significantly higher in the test group than in the control and naive groups (P-value <0.05), while Zn levels were higher in the test group than in the control group with no significant difference. Serum glutamicoxaloacetic transaminase concentrations in the test group were significantly lower than those in other groups (P-value <0.05), while serum glutamate pyruvic transaminase concentrations were significantly higher in test compared with those in other groups (P-value <0.05). Moreover, alkaline phosphatase in the control and test groups were significantly lower than that in the naive group (P-value <0.05). Conclusion: TNG treatment increased Zn and Cu levels and thus increased resistance to Leishmania because of the role of Zn and Cu; therefore, TNG therapy will be useful for treating cutaneous leishmania. In addition, the decrease of serum glutamicoxaloacetic transaminase activity can be an index of therapeutic process of TNG. PMID:25945241

  6. Role of STAT3 in Angiotensin II-Induced Hypertension and Cardiac Remodeling Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation

    PubMed Central

    Zouein, Fouad A.; Zgheib, Carlos; Hamza, Shereen; Fuseler, John W.; Hall, John E.; Soljancic, Andrea; Lopez-Ruiz, Arnaldo; Kurdi, Mazen; Booz, George W.

    2013-01-01

    STAT3 is involved in protection of the heart provided by ischemic preconditioning. However, the role of this transcription factor in the heart in chronic stresses such as hypertension has not been defined. We assessed whether STAT3 is important in hypertension-induced cardiac remodeling using mice with reduced STAT3 activity due to a S727A mutation (SA/SA). Wild type (WT) and SA/SA mice received angiotensin (ANG) II or saline for 17 days. ANG II increased mean arterial and systolic pressure in SA/SA and WT mice, but cardiac levels of cytokines associated with heart failure were increased less in SA/SA mice. Unlike WT mice, hearts of SA/SA mice showed signs of developing systolic dysfunction as evidenced by reduction in ejection fraction and fractional shortening. In the left ventricle of both WT and SA/SA mice, ANG II induced fibrosis. However, fibrosis in SA/SA mice appeared more extensive and was associated with loss of myocytes. Cardiac hypertrophy as indexed by heart to body weight ratio and left ventricular anterior wall dimension during diastole was greater in WT mice. In WT+ANG II mice there was an increase in the mass of individual myofibrils. In contrast, cardiac myocytes of SA/SA+ANG II mice showed a loss in myofibrils and myofibrillar mass density was decreased during ANG II infusion. Our findings reveal that STAT3 transcriptional activity is important for normal cardiac myocyte myofibril morphology. Loss of STAT3 may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure. PMID:23364341

  7. Immune response of BALB/c mice against an experimental vaccine of Alum precipitated autoclaved Leishmania major (Alum-ALM) mixed with BCG or Mycobacterium vaccae.

    PubMed

    Nateghi Rostami, M; Keshavarz, H; Khamesipour, A

    2010-04-01

    Immune response in BALB/c mice immunized 3 times with different doses (50 μg or 200 μg of protein) of Alum precipitated autoclaved Leishmania major (Alum-ALM) mixed with either BCG (1x10(7); CFU) or different doses of killed Mycobacterium vaccae (1x10(6), 1x10(7)) was assessed. Mice immunized with low dose of Alum-ALM mixed with either BCG or low M. vaccae showed a significantly higher IFN-gamma production and a lower IL-4 level and a significantly lower parasite burden compared to the control PBS injected group. It seems that immunization with a low dose of Alum-ALM mixed with an adjuvant induces a Th1 type of immune response in susceptible BALB/c mice.

  8. Combination therapy using Pentostam and Praziquantel improves lesion healing and parasite resolution in BALB/c mice co-infected with Leishmania major and Schistosoma mansoni

    PubMed Central

    2013-01-01

    Background Most natural host populations are exposed to a diversity of parasite communities and co-infection of hosts by multiple parasites is commonplace across a diverse range of systems. Co-infection with Leishmania major and Schistosoma mansoni may have important consequences for disease development, severity and transmission dynamics. Pentavalent antimonials and Praziquantel (PZQ) have been relied upon as a first line of treatment for Leishmania and Schistosoma infections respectively. However, it is not clear how combined therapy with the standard drugs will affect the host and parasite burden in concomitance. The aim of the current study was to determine the efficacy of combined chemotherapy using Pentostam and PZQ in BALB/c mice co-infected with L. major and S. mansoni. Methods The study used BALB/c mice infected with L. major and S. mansoni. A 3 × 4 factorial design with three parasite infection groups (Lm, Sm, Lm + Sm designated as groups infected with L. major, S. mansoni and L. major + S. mansoni, respectively) and four treatment regimens [P, PZQ, P + PZQ and PBS designating Pentostam®(GlaxoSmithKline UK), Praziquantel (Biltricide®, Bayer Ag. Leverkusen, Germany), Pentostam + Praziquantel and Phosphate buffered saline] as factors was applied. In each treatment group, there were 10 mice. Lesion development was monitored for 10 weeks. The parasite load, body weight, weight of the spleen and liver were determined between week 8 and week 10. Results Chemotherapy using the first line of treatment for L. major and S. mansoni reduced the lesion size and parasite loads but did not affect the growth response, spleen and liver. In the co-infected BALB/c mice, the use of Pentostam or PZQ did not result in any appreciable disease management. However, treatment with P + PZQ resulted in significantly (p < 0.05) larger reduction of lesions, net increase in the body weight, no changes in the spleen and liver weight and reduced Leishman

  9. Profiling Trait Anxiety: Transcriptome Analysis Reveals Cathepsin B (Ctsb) as a Novel Candidate Gene for Emotionality in Mice

    PubMed Central

    Czibere, Ludwig; Baur, Laura A.; Wittmann, Anke; Gemmeke, Katja; Steiner, Andrea; Weber, Peter; Pütz, Benno; Ahmad, Nafees; Bunck, Mirjam; Graf, Cornelia; Widner, Regina; Kühne, Claudia; Panhuysen, Markus; Hambsch, Boris; Rieder, Gabriele; Reinheckel, Thomas; Peters, Christoph; Holsboer, Florian; Landgraf, Rainer; Deussing, Jan M.

    2011-01-01

    Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait “anxiety”. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4). Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy. PMID:21897848

  10. Aspartame and the hippocampus: Revealing a bi-directional, dose/time-dependent behavioural and morphological shift in mice.

    PubMed

    Onaolapo, Adejoke Y; Onaolapo, Olakunle J; Nwoha, Polycarp U

    2017-03-01

    Changes, in behaviour, oxidative markers of stress and hippocampal morphology were evaluated following aspartame administration. Mice, (20-22g each) were given vehicle (10ml/kg) or aspartame (20, 40, 80 and 160mg/kg) daily for 28days. They were tested in the Y-maze, radial-arm maze and elevated plus-maze (EPM) after the first and last dose of vehicle or aspartame; and then sacrificed. Hippocampal slices were analysed for aspartic acid, nitric oxide (NO) and superoxide dismutase (SOD); and processed for general histology and neuritic plaques. Glial fibrillary-acid protein (GFAP) expression and neuron-specific enolase (NSE) activities were determined. Radial-arm maze scores increased significantly after acute administration at 80 and 160mg/kg. Repeated administration at 20 and 40mg/kg (Y-maze) and at 40mg/kg (radial-arm maze) was also associated with increased scores, however, performance decreased at higher doses. EPM tests revealed anxiogenic responses following both acute and repeated administration. Significant increase in SOD and NO activities were observed at 40, 80 and 160mg/kg. Neuron counts reduced at higher doses of aspartame. At 40, 80 and 160mg/kg, fewer GFAP-reactive astrocytes were observed in the cornus ammonis, but increased GFAP-reactivity was observed in the dentate gyrus subgranular zone. NSE-positive neurons were readily identifiable within the dentate gyrus at the lower doses of aspartame; but at 160mg/kg, there was marked neuron loss and reduction in NSE-positive neurons. Oral aspartame significantly altered behaviour, anti-oxidant status and morphology of the hippocampus in mice; also, it may probably trigger hippocampal adult neurogenesis.

  11. Gene Expression Profiling of Astrocytes from Hyperammonemic Mice Reveals Altered Pathways for Water and Potassium Homeostasis In Vivo

    PubMed Central

    LICHTER-KONECKI, UTA; MANGIN, JEAN MARIE; GORDISH-DRESSMAN, HEATHER; HOFFMAN, ERIC P.; GALLO, VITTORIO

    2014-01-01

    Acute hyperammonemia (HA) causes cerebral edema and brain damage in children with urea cycle disorders (UCDs) and in patients in acute liver failure. Chronic HA is associated with developmental delay and mental retardation in children with UCDs, and with neuropsychiatric symptoms in patients with chronic liver failure. Astrocytes are a major cellular target of hyperammonemic encephalopathy, and changes occurring in these cells are thought to be causally related to the brain edema of acute HA. To study the effect of HA on astrocytes in vivo, we crossed the Otcspf mouse, a mouse with the X-linked UCD ornithine transcarbamylase (OTC) deficiency, with the hGFAP-EGFP mouse, a mouse selectively expressing green fluorescent protein in astrocytes. We used FACS to purify astrocytes from the brains of hyperammonemic and healthy Otcspf/GFAP-EGFP mice. RNA isolated from these astrocytes was used in microarray expression analyses and qRT-PCR. When compared with healthy littermates, we observed a significant downregulation of the gap-junction channel connexin 43 (Cx43) the water channel aquaporin 4 (Aqp4) genes, and the astrocytic inward-rectifying potassium channel (Kir) genes Kir4.1 and Kir5.1 in hyperammonemic mice. Aqp4, Cx43, and Kir4.1/ Kir5.1 are co-localized to astrocytic end-feet at the brain vasculature, where they regulate potassium and water transport. Since, NH4+ ions can permeate water and K+-channels, downregulation of these three channels may be a direct effect of elevated blood ammonia levels. Our results suggest that alterations in astrocyte-mediated water and potassium homeostasis in brain may be key to the development of the brain edema. PMID:18186079

  12. Lipidomic analysis of the liver identifies changes of major and minor lipid species in adiponectin deficient mice

    PubMed Central

    Wanninger, Josef; Liebisch, Gerhard; Schmitz, Gerd; Bauer, Sabrina; Eisinger, Kristina; Neumeier, Markus; Ouchi, Noriyuki; Walsh, Kenneth; Buechler, Christa

    2014-01-01

    Adiponectin protects from hepatic fat storage but adiponectin deficient mice (APN−/−) fed a standard chow do not develop liver steatosis. This indicates that other pathways might be activated to compensate for adiponectin deficiency. An unbiased and comprehensive screen was performed to identify hepatic alterations of lipid classes in these mice. APN−/− mice had decreased hepatic cholesteryl esters while active SREBP2 and systemic total cholesterol were not altered. Upregulation of cytochromes for bile acid synthesis suggests enhanced biliary cholesterol excretion. Analysis of 37 individual fatty acid species showed reduced stearate whereas total fatty acids were not altered. Total amount of triglycerides and phospholipids were equally abundant. A selective increase of monounsaturated phosphatidylcholine and phosphatidylethanolamine which positively correlate with hepatic and systemic triglycerides with the latter being elevated in APN−/− mice, was identified. Stearoyl-CoA desaturase 1 (SCD1) is involved in the synthesis of monounsaturated fatty acids and despite higher mRNA expression enzyme activity was not enhanced. Glucosylceramide postulated to contribute to liver damage was decreased. This study demonstrates that adiponectin deficiency is associated with hepatic changes in lipid classes in mice fed a standard chow which may protect from liver steatosis. PMID:22465357

  13. Induction of transplantation tolerance in mice across major histocompatibility barrier by using allogeneic thymus transplantation and total lymphoid irradiation

    SciTech Connect

    Waer, M.; Palathumpat, V.; Sobis, H.; Vandeputte, M. )

    1990-07-15

    The use of allogeneic thymus transplantation as a means of inducing tolerance across MHC barriers was investigated in thymectomized, total lymphoid irradiated BALB/c mice. In 90% of the animals long term outgrowth of histologically normal C57BL thymus grafts was observed. None of the latter animals was chimeric. All thymus graft-bearing mice showed specific nonresponsiveness for C57BL MHC Ag in mixed lymphocyte reaction and cell-mediated lympholysis. Spleen cells of the C57BL thymus-bearing mice were unable to induce lethal graft-vs-host disease in neonatal (BALB/c X C57BL) F1 mice but provoked a vigorous graft-vs-host disease reaction in (BALB/c x C3H) F1 neonates. Tolerant mice permanently accepted C57BL heart and pancreas grafts, but all rejected C3H grafts. Induction of tolerance of BALB/c pre-T cells through allogeneic thymus graft and/or specific suppressor cells seems to be involved. The present model offers new opportunities to study thymocyte maturation in a fully allogeneic environment and may yield applications for clinical organ transplantation.

  14. Inhibition of the multiplication of Mycobacterium leprae by vaccination with a recombinant M. bovis BCG strain that secretes major membrane protein II in mice.

    PubMed

    Maeda, Y; Tamura, T; Matsuoka, M; Makino, M

    2009-10-01

    The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-gamma) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8(+) T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-gamma on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.

  15. Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

    PubMed Central

    Rivera, Mercedes F.; Lee, Ju-Youn; Aneja, Monika; Goswami, Vishalkant; Liu, Liying; Velsko, Irina M.; Chukkapalli, Sasanka S.; Bhattacharyya, Indraneel; Chen, Hao; Lucas, Alexandra R.; Kesavalu, Lakshmyya N.

    2013-01-01

    Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoEnull) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoEnull mice. PMID:23451182

  16. Sphingomyelin Liposomes Containing Soluble Leishmania major antigens Induced Strong Th2 Immune Response in BALB/c Mice

    PubMed Central

    Chavoshian, Omid; Biari, Nazanin; Badiee, Ali; Khamesipour, Ali; Abbasi, Azam; Saberi, Zahra; Jalali, Seyed Amir; Jaafari, Mahmoud Reza

    2013-01-01

    Objective(s): Soluble Leishmania antigens (SLA) provide suitable protection against leishmaniasis in murine model when delivered by an appropriate delivery system. Liposomes have been shown to be suitable vaccine delivery systems against leishmaniasis, however, the phospholipase-A (PLA) activity of SLA is a drawback to prepare a stable liposomal SLA. One strategy to overcome this problem might be using a lipid which is resistant to PLA activity of SLA such as sphingomyelin (SM). The aim of this study was to evaluate the effect of stable SM liposomes containing SLA on the immune response induced against leishmaniasis in BALB/c mice . Materials and Methods: BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with SLA, SM-liposome-SLA, empty liposome or buffer. As criteria for protection, footpads swelling at the site of challenge and foot parasite loads were assessed. The immune responses were also evaluated by determination of IgG subtypes and the level of IFN-γ and IL-4 in cultured splenocytes. Results: The group of mice receiving SM-liposome-SLA, showed a significant large footpad swelling, higher parasite burden in foot and higher IL-4 level compared to the group immunized with buffer. In terms of IgG and IgG isotypes, there was no significant difference between the mice receiving SM-liposome-SLA and the mice that received buffer. Moreover, the immune response induced by SM-liposome-SLA showed no significant difference compared with the one caused by SLA alone. Conclusion: It is concluded that SM-liposome-SLA is not an appropriate strategy to induce Th1 immune response and protect the mice against Leishmaniasis; however, SM-liposomes could be suitable vaccine delivery systems when a Th2 response is needed. PMID:24171074

  17. Endogenous oncornaviral gene expression in adult and fetal mice: quantitative, histologic, and physiologic studies of the major viral glycorprotein, gp70

    PubMed Central

    1976-01-01

    Endogenous expression of the murine leukemia virus (MuLV) genome has been studied in a number of strains of mice. Expression of the major envelope glycoprotein, gp70, is restricted to certain anatomical sites and cell types, prominent among which are lymphoid and epithelial cells. On a quantitative basis, the major site of gp70 expression is the male genital tract. During development, gp70 first appears in the hematopoietic liver of 14-day-old embryos and by day 18, it is already expressed at anatomical sites similar to those of the adult. In toto, these results show that control of expression of the MuLV genome in adult and developing mice is linked to differentiation. PMID:172586

  18. Gene expression profiling of R6/2 transgenic mice with different CAG repeat lengths reveals genes associated with disease onset and progression in Huntington's disease.

    PubMed

    Tang, Bin; Seredenina, Tamara; Coppola, Giovanni; Kuhn, Alexandre; Geschwind, Daniel H; Luthi-Carter, Ruth; Thomas, Elizabeth A

    2011-06-01

    R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice

  19. In vivo metabolism of L-methionine in mice: evidence for stereoselective formation of methionine-d-sulfoxide and quantitation of other major metabolites.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2006-12-01

    Flavin-containing monooxygenases (FMOs) 1-4 oxidize methionine (Met) to methionine sulfoxide (MetO). FMO3, the primary isoform expressed in adult human liver, has the lowest Km and favors methionine-d-sulfoxide (Met-d-O) formation over methionine-l-sulfoxide. Because female mice, but not males, also express FMO3 in liver, levels of Met and its major metabolites were determined in male or female mice dosed with 400 mg/kg Met i.p. The results show that Met levels in male and female mouse liver or plasma increased significantly at both 15 and 30 min after the Met treatment; Met plasma and liver levels at 30 min were similar to or lower than the corresponding levels at 15 min. Liver and plasma MetO levels increased significantly in both sexes at 30 min, and Met-d-O was the major MetO diastereomer detected. Interestingly, less than 0.1% of the Met dose was excreted in the urine (0-24 h) as Met and Met-d-O. S-Adenosylmethionine (SAM) was the major metabolite detected in liver at 15 min. Liver SAM levels at 30 min were lower than the levels at 15 min, and the plasma SAM levels at both 15 and 30 min were much lower than the corresponding levels in the liver. Increases in liver and/or plasma S-adenosyl-l-homocysteine, 5'-deoxy-5'-(methylthio)adenosine, and N-acetyl-l-methionine were also detected. Taken together, these results suggest that mice extensively and rapidly used the Met dose. Although mice exhibited increases in tissue MetO levels, a major role for FMO3 in Met-d-O formation is not certain since the MetO increases were mostly similar in both males and females.

  20. Molecular Phenotyping of Immune Cells from Young NOD Mice Reveals Abnormal Metabolic Pathways in the Early Induction Phase of Autoimmune Diabetes

    PubMed Central

    Wu, Jian; Kakoola, Dorothy N.; Lenchik, Nataliya I.; Desiderio, Dominic M.; Marshall, Dana R.; Gerling, Ivan C.

    2012-01-01

    Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse – a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (∼90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes. PMID:23071669

  1. Proteomic Analysis of Post-synaptic Density Fractions from Shank3 Mutant Mice Reveals Brain Region Specific Changes Relevant to Autism Spectrum Disorder

    PubMed Central

    Reim, Dominik; Distler, Ute; Halbedl, Sonja; Verpelli, Chiara; Sala, Carlo; Bockmann, Juergen; Tenzer, Stefan; Boeckers, Tobias M.; Schmeisser, Michael J.

    2017-01-01

    Disruption of the human SHANK3 gene can cause several neuropsychiatric disease entities including Phelan-McDermid syndrome, autism spectrum disorder (ASD), and intellectual disability. Although, a wide array of neurobiological studies strongly supports a major role for SHANK3 in organizing the post-synaptic protein scaffold, the molecular processes at synapses of individuals harboring SHANK3 mutations are still far from being understood. In this study, we biochemically isolated the post-synaptic density (PSD) fraction from striatum and hippocampus of adult Shank3Δ11-/- mutant mice and performed ion-mobility enhanced data-independent label-free LC–MS/MS to obtain the corresponding PSD proteomes (Data are available via ProteomeXchange with identifier PXD005192). This unbiased approach to identify molecular disturbances at Shank3 mutant PSDs revealed hitherto unknown brain region specific alterations including a striatal decrease of several molecules encoded by ASD susceptibility genes such as the serine/threonine kinase Cdkl5 and the potassium channel KCa1.1. Being the first comprehensive analysis of brain region specific PSD proteomes from a Shank3 mutant line, our study provides crucial information on molecular alterations that could foster translational treatment studies for SHANK3 mutation-associated synaptopathies and possibly also ASD in general. PMID:28261056

  2. Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice.

    PubMed

    Yu, Hong; Karunakaran, Karuna P; Jiang, Xiaozhou; Brunham, Robert C

    2014-08-06

    An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Paleogenetic Analyses Reveal Unsuspected Phylogenetic Affinities between Mice and the Extinct Malpaisomys insularis, an Endemic Rodent of the Canaries

    PubMed Central

    Gros-Balthazard, Muriel; Hughes, Sandrine; Alcover, Josep Antoni; Hutterer, Rainer; Rando, Juan Carlos; Michaux, Jacques; Hänni, Catherine

    2012-01-01

    Background The lava mouse, Malpaisomys insularis, was endemic to the Eastern Canary islands and became extinct at the beginning of the 14th century when the Europeans reached the archipelago. Studies to determine Malpaisomys' phylogenetic affinities, based on morphological characters, remained inconclusive because morphological changes experienced by this insular rodent make phylogenetic investigations a real challenge. Over 20 years since its first description, Malpaisomys' phylogenetic position remains enigmatic. Methodology/Principal Findings In this study, we resolved this issue using molecular characters. Mitochondrial and nuclear markers were successfully amplified from subfossils of three lava mouse samples. Molecular phylogenetic reconstructions revealed, without any ambiguity, unsuspected relationships between Malpaisomys and extant mice (genus Mus, Murinae). Moreover, through molecular dating we estimated the origin of the Malpaisomys/mouse clade at 6.9 Ma, corresponding to the maximal age at which the archipelago was colonised by the Malpaisomys ancestor via natural rafting. Conclusion/Significance This study reconsiders the derived morphological characters of Malpaisomys in light of this unexpected molecular finding. To reconcile molecular and morphological data, we propose to consider Malpaisomys insularis as an insular lineage of mouse. PMID:22363563

  4. Changes in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography

    NASA Astrophysics Data System (ADS)

    Kerckhofs, G.; Durand, M.; Vangoitsenhoven, R.; Marin, C.; van der Schueren, B.; Carmeliet, G.; Luyten, F. P.; Geris, L.; Vandamme, K.

    2016-10-01

    High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 μm scale, no change was observed in the intracortical structure. At 1 μm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging.

  5. Changes in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography

    PubMed Central

    Kerckhofs, G.; Durand, M.; Vangoitsenhoven, R.; Marin, C.; Van der Schueren, B.; Carmeliet, G.; Luyten, F. P.; Geris, L.; Vandamme, K.

    2016-01-01

    High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 μm scale, no change was observed in the intracortical structure. At 1 μm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging. PMID:27759061

  6. Changes in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography.

    PubMed

    Kerckhofs, G; Durand, M; Vangoitsenhoven, R; Marin, C; Van der Schueren, B; Carmeliet, G; Luyten, F P; Geris, L; Vandamme, K

    2016-10-19

    High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 μm scale, no change was observed in the intracortical structure. At 1 μm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging.

  7. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice

    PubMed Central

    Shiao, Young-Ji; Liou, Kuo-Tong; Hsu, Wei-Hsiang; Hsieh, Pei-Hsuan; Lee, Chi-Ying; Chen, Yet-Ran; Lin, Yun-Lian

    2015-01-01

    Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood–brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke. PMID:26492191

  8. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

    PubMed

    Chen, Hong-Jhang; Shen, Yuh-Chiang; Shiao, Young-Ji; Liou, Kuo-Tong; Hsu, Wei-Hsiang; Hsieh, Pei-Hsuan; Lee, Chi-Ying; Chen, Yet-Ran; Lin, Yun-Lian

    2015-01-01

    Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.

  9. Gastric mucosal hyperplasia via upregulation of gastrin induced by persistent activation of gastric innate immunity in major histocompatibility complex class II deficient mice

    PubMed Central

    Fukui, T; Nishio, A; Okazaki, K; Uza, N; Ueno, S; Kido, M; Inoue, S; Kitamura, H; Kiriya, K; Ohashi, S; Asada, M; Tamaki, H; Matsuura, M; Kawasaki, K; Suzuki, K; Uchida, K; Fukui, H; Nakase, H; Watanabe, N; Chiba, T

    2006-01-01

    Background and aim Major histocompatibility complex class II deficient (Aα0/0) mice have decreased CD4+ T cells, making them immunologically similar to patients with acquired immunodeficiency syndrome (AIDS). Both patients with AIDS and Aα0/0 mice have hypertrophic gastric folds. To clarify the mechanism of gastric mucosal hyperplasia, we investigated the pathophysiology and the role of the innate immunity in the stomach of Aα0/0 mice. Methods Stomachs from 1–6 month old Aα0/0 mice, kept under specific pathogen free conditions, were examined at 1 month intervals histologically and immunohistochemically. Gene expression of proinflammatory cytokines, Toll‐like receptors (TLRs), cyclooxygenase (COX)‐2, and myeloperoxidase (MPO) activity in the gastric mucosa was investigated. Serum gastrin levels and gastric acidity were measured. Bacterial culture of the stomach was performed. To clarify the roles of hypergastrinaemia in the gastric mucosa, a gastrin receptor antagonist (AG041R) was administered. Results Aα0/0 mice had a diffusely thick corpus mucosa with infiltration of CD11b+ granulocytes and macrophages. Anti‐Ki67 staining demonstrated expansion of the proliferating neck zone. Gene expression of interleukin 1β, interferon γ, TLR‐2, TLR‐4, and COX‐2 were upregulated, and MPO activity was increased. Only a small amount of non‐pathogenic bacteria was detected in the stomach. Serum gastrin levels and Reg‐Iα positive cells in the gastric mucosa increased, despite normal gastric acidity. After treatment with AG041R, gastric mucosal thickness was significantly reduced. Conclusion Persistent activation of innate immunity in the stomach induced gastric mucosal hyperplasia through upregulation of gastrin synthesis in Aα0/0 mice, suggesting a pathophysiology similar to the gastric changes in patients with AIDS. PMID:16322110

  10. Carnosic acid as a major bioactive component in rosemary extract ameliorates high-fat-diet-induced obesity and metabolic syndrome in mice.

    PubMed

    Zhao, Yantao; Sedighi, Rashin; Wang, Pei; Chen, Huadong; Zhu, Yingdong; Sang, Shengmin

    2015-05-20

    In this study, we investigated the preventive effects of carnosic acid (CA) as a major bioactive component in rosemary extract (RE) on high-fat-diet-induced obesity and metabolic syndrome in mice. The mice were given a low-fat diet, a high-fat diet or a high-fat diet supplemented with either 0.14% or 0.28% (w/w) CA-enriched RE (containing 80% CA, RE#1L and RE#1H), or 0.5% (w/w) RE (containing 45% CA, RE#2), for a period of 16 weeks. There was the same CA content in the RE#1H and RE#2 diets and half of this amount in the RE#1L diet. The dietary RE supplementation significantly reduced body weight gain, percent of fat, plasma ALT, AST, glucose, insulin levels, liver weight, liver triglyceride, and free fatty acid levels in comparison with the mice fed with a HF diet without RE treatment. RE administration also decreased the levels of plasma and liver malondialdehyde, advanced glycation end products (AGEs), and the liver expression of receptor for AGE (RAGE) in comparison with those for mice of the HF group. Histological analyses of liver samples showed decreased lipid accumulation in hepatocytes in mice administrated with RE in comparison with that of HF-diet-fed mice. Meanwhile, RE administration enhanced fecal lipid excretion to inhibit lipid absorption and increased the liver GSH/GSSG ratio to perform antioxidant activity compared with HF group. Our results demonstrate that rosemary is a promising dietary agent to reduce the risk of obesity and metabolic syndrome.

  11. 2-D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents.

    PubMed

    Schmidt, Hendrik; Gelhaus, Christoph; Latendorf, Ties; Nebendahl, Melanie; Petersen, Arnd; Krause, Susanne; Leippe, Matthias; Becker, Wolf-Meinhard; Janssen, Ottmar

    2009-07-01

    Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut-derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre-packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut-derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia-type and Indonesian peanuts were compared by 1- and 2-DE. We identified more than hundred individual components in these extracts by MS and provide a high-resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.

  12. Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair

    PubMed Central

    Poobalasingam, Thanushiyan; Yates, Laura L.; Walker, Simone A.; Pereira, Miguel; Gross, Nina Y.; Ali, Akmol; Kolatsi-Joannou, Maria; Jarvelin, Marjo-Riitta; Pekkanen, Juha; Papakrivopoulou, Eugenia; Long, David A.; Griffiths, Mark; Wagner, Darcy; Königshoff, Melanie; Hind, Matthew; Minelli, Cosetta; Lloyd, Clare M.

    2017-01-01

    ABSTRACT Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung

  13. Heterozygous Vangl2(Looptail) mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair.

    PubMed

    Poobalasingam, Thanushiyan; Yates, Laura L; Walker, Simone A; Pereira, Miguel; Gross, Nina Y; Ali, Akmol; Kolatsi-Joannou, Maria; Jarvelin, Marjo-Riitta; Pekkanen, Juha; Papakrivopoulou, Eugenia; Long, David A; Griffiths, Mark; Wagner, Darcy; Königshoff, Melanie; Hind, Matthew; Minelli, Cosetta; Lloyd, Clare M; Dean, Charlotte H

    2017-02-24

    Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as Idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive pulmonary Disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising.The Planar Cell Polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly due to the perinatal lethality of many PCP mouse mutant lines.Here we have investigated heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2(Lp/+) lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin modifying protein cofilin. In addition, we show that Vangl2(Lp/+) lungs exhibit many of the hallmarks of tissue damage including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in the lung disease, emphysema.In vitro, VANGL2 disruption impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking (a tissue damaging insult) on lung function. Finally, we show that that PCP genes VANGL2 and

  14. Reduced glycine transporter type 1 expression leads to major changes in glutamatergic neurotransmission of CA1 hippocampal neurones in mice

    PubMed Central

    Martina, Marzia; Turcotte, Marie-Eve B; Halman, Samantha; Tsai, Guochuan; Tiberi, Mario; Coyle, Joseph T; Bergeron, Richard

    2005-01-01

    To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral–CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/−) as compared to wild type (WT) littermates using whole-cell patch-clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc-induced antagonism in N-methyl-d-aspartate receptor (NMDAR) currents of GlyT1+/− mice. Moreover, the ratio α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission. PMID:15661817

  15. Danshensu, a major water-soluble component of Salvia miltiorrhiza, enhances the radioresponse for Lewis Lung Carcinoma xenografts in mice

    PubMed Central

    Cao, Hong-Ying; Ding, Rui-Lin; Li, Meng; Yang, Mao-Nan; Yang, Ling-Lin; Wu, Jing-Bo; Yang, Bo; Wang, Jing; Luo, Cui-Lian; Wen, Qing-Lian

    2017-01-01

    The molecule 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid (danshensu), a herbal preparation used in traditional Chinese medicine, has been found to possess potential antitumor and anti-angiogenesis effects. The aim of the present study was to investigate the efficacy of the combination of radiation therapy (RT) with danshensu in the treatment of Lewis lung carcinoma (LLC) xenografts, whilst exploring and evaluating the mechanism involved. In total, 8-week old female C57BL/6J mice were randomly assigned into 3 groups to receive: RT, RT + cisplatin and RT + danshensu, respectively, when LLC reached 100–150 mm3. Each group was divided into 7 subgroups according to the different irradiation doses that were administered. Tumor growth curves were created and the sensitization enhancement ratios of the drugs were calculated. The experiment was then repeated, and the 4 groups of tumor-bearing mice were treated with natural saline, danshensu, RT + danshensu and RT, respectively. The mice were sacrificed on day 7, and tumor tissue and blood were collected to determine microvessel density, the expression of proangiogenic factors, and the levels of blood thromboxane B2 and 6-keto-prostaglandin-F1α. Tumor hypoxia was also detected using in vivo fluorescence imaging. With respect to LLC xenografts, treatment with danshensu + RT significantly enhanced the effects of tumor growth inhibition (P<0.05). Furthermore, tumor vasculature was remodeled and microcirculation was improved, which significantly reduced tumor hypoxia (P<0.05). The present study demonstrated that danshensu significantly enhanced the radioresponse of LLC xenografts in mice. The mechanism involved may be associated with the alleviation of tumor cell hypoxia following treatment with danshensu + RT, caused by the improvement of tumor microcirculation and the remodeling of tumor vasculature. PMID:28356936

  16. Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein.

    PubMed

    Zalckvar, Einat; Paulus, Christina; Tillo, Desiree; Asbach-Nitzsche, Alexandra; Lubling, Yaniv; Winterling, Carla; Strieder, Nicholas; Mücke, Katrin; Goodrum, Felicia; Segal, Eran; Nevels, Michael

    2013-08-06

    Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.

  17. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

    PubMed Central

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D.; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L.; Gapin, Laurent; Marrack, Philippa; Kappler, John W.

    2016-01-01

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  18. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  19. Comparative analysis of single and combined APP/APLP knockouts reveals reduced spine density in APP-KO mice that is prevented by APPsα expression

    PubMed Central

    2014-01-01

    Synaptic dysfunction and synapse loss are key features of Alzheimer’s pathogenesis. Previously, we showed an essential function of APP and APLP2 for synaptic plasticity, learning and memory. Here, we used organotypic hippocampal cultures to investigate the specific role(s) of APP family members and their fragments for dendritic complexity and spine formation of principal neurons within the hippocampus. Whereas CA1 neurons from APLP1-KO or APLP2-KO mice showed normal neuronal morphology and spine density, APP-KO mice revealed a highly reduced dendritic complexity in mid-apical dendrites. Despite unaltered morphology of APLP2-KO neurons, combined APP/APLP2-DKO mutants showed an additional branching defect in proximal apical dendrites, indicating redundancy and a combined function of APP and APLP2 for dendritic architecture. Remarkably, APP-KO neurons showed a pronounced decrease in spine density and reductions in the number of mushroom spines. No further decrease in spine density, however, was detectable in APP/APLP2-DKO mice. Mechanistically, using APPsα-KI mice lacking transmembrane APP and expressing solely the secreted APPsα fragment we demonstrate that APPsα expression alone is sufficient to prevent the defects in spine density observed in APP-KO mice. Collectively, these studies reveal a combined role of APP and APLP2 for dendritic architecture and a unique function of secreted APPs for spine density. PMID:24684730

  20. Whole genome, whole population sequencing reveals that loss of signaling networks is the major adaptive strategy in a constant environment.

    PubMed

    Kvitek, Daniel J; Sherlock, Gavin

    2013-11-01

    Molecular signaling networks are ubiquitous across life and likely evolved to allow organisms to sense and respond to environmental change in dynamic environments. Few examples exist regarding the dispensability of signaling networks, and it remains unclear whether they are an essential feature of a highly adapted biological system. Here, we show that signaling network function carries a fitness cost in yeast evolving in a constant environment. We performed whole-genome, whole-population Illumina sequencing on replicate evolution experiments and find the major theme of adaptive evolution in a constant environment is the disruption of signaling networks responsible for regulating the response to environmental perturbations. Over half of all identified mutations occurred in three major signaling networks that regulate growth control: glucose signaling, Ras/cAMP/PKA and HOG. This results in a loss of environmental sensitivity that is reproducible across experiments. However, adaptive clones show reduced viability under starvation conditions, demonstrating an evolutionary tradeoff. These mutations are beneficial in an environment with a constant and predictable nutrient supply, likely because they result in constitutive growth, but reduce fitness in an environment where nutrient supply is not constant. Our results are a clear example of the myopic nature of evolution: a loss of environmental sensitivity in a constant environment is adaptive in the short term, but maladaptive should the environment change.

  1. Hormone profiling and transcription analysis reveal a major role of ABA in tomato salt tolerance.

    PubMed

    Yang, Rongchao; Yang, Ting; Zhang, Haijun; Qi, Yan; Xing, Yanxia; Zhang, Na; Li, Ren; Weeda, Sarah; Ren, Shuxin; Ouyang, Bo; Guo, Yang-Dong

    2014-04-01

    The response and adaptation of plants to different environmental stresses are of great interest as they provide the key to understanding the mechanisms underlying stress tolerance. In this study, the changing patterns of four endogenous hormones and various physiological and biochemical parameters of both a salt-tolerant (LA2711) and a salt-sensitive (ZS-5) tomato cultivar were examined under salt stress and non-stress conditions. Additionally, the transcription of key genes in the abscisic acid (ABA) biosynthesis and metabolism were analyzed at different time points. The results indicated that gene expression responsible for ABA biosynthesis and metabolism coincided with the hormone level, and SlNCED1 and SlCYP707A3 may play major roles in the process. LA2711 performed superior to ZS-5 on various parameters, including seed germination, Na(+) compartmentation, selective absorption of K(+), and antioxidant enzymes activity. The difference in salt tolerance between the two genotypes could be attributed to the different levels of ABA due to differences in gene expression of key genes in ABA biosynthesis and metabolism. Although gibberellin, cytokinin and auxin were involved, our results indicated that ABA signaling plays a major role in tomato salt tolerance. As compared to ZS-5, LA2711 had a higher capability to selectively absorb and redistribute K(+) and a higher tolerance to Na(+) in young leaves, which may be the main physiological mechanisms of salt tolerance.

  2. Effect of CoCl(2) treatment on major and trace elements metabolism and protein concentration in mice.

    PubMed

    Zaksas, Nataliya; Gluhcheva, Yordanka; Sedykh, Sergey; Madzharova, Maria; Atanassova, Nina; Nevinsky, Georgy

    2013-01-01

    Cobalt (Co) is a transition metal and an essential trace element, required for vitamin B(12) biosynthesis, enzyme activation and other biological processes, but toxic in high concentrations. There is lack of data for the effect of long-term Co(II) treatment on the concentrations of other trace elements. We estimate the influence of cobalt chloride (CoCl(2)) on the relative content of different metals in mouse plasma using two-jet arc plasmatron atomic emission and on the total protein content. On average, the content of different elements in the plasma of 2-month-old balb/c mice (control group) decreased in the order: Ca>Mg>Si>Fe>Zn>Cu≥Al≥B. The treatment of mice for 60 days with CoCl(2) (daily dose 125 mg/kg) did not appreciably change the relative content of Ca, Cu, and Zn, while a 2.4-fold statistically significant decrease in the content of B and significant increase in the content of Mg (1.4-fold), Al and Fe (2.0-fold) and Si (3.2-fold) was found. A detectable amount of Mo was observed only for two control mice, while the plasma of 9 out of 16 mice of the treated group contained this metal. The administration of Co made its concentration detectable in the plasma of all mice of the treated group, but the relative content varied significantly. The treatment led to a 2.2-fold decrease in the concentration of the total plasma protein. Chronic exposure to CoCl(2) affects homeostasis as well as the concentrations and metabolism of other essential elements, probably due to competition of Co ions for similar binding sites within cells, altered signal transduction and protein biosynthesis. Long-term treatment also leads to significant weight changes and reduces the total protein concentration. The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions and different physiological processes. Copyright © 2012 Elsevier GmbH. All rights reserved.

  3. Deletion of the Androgen Receptor in Adipose Tissue in Male Mice Elevates Retinol Binding Protein 4 and Reveals Independent Effects on Visceral Fat Mass and on Glucose Homeostasis

    PubMed Central

    McInnes, Kerry J.; Smith, Lee B.; Hunger, Nicole I.; Saunders, Philippa T.K.; Andrew, Ruth; Walker, Brian R.

    2012-01-01

    Testosterone deficiency is epidemic in obese ageing males with type 2 diabetes, but the direction of causality remains unclear. Testosterone-deficient males and global androgen receptor (AR) knockout mice are insulin resistant with increased fat, but it is unclear whether AR signaling in adipose tissue mediates body fat redistribution and alters glucose homoeostasis. To investigate this, mice with selective knockdown of AR in adipocytes (fARKO) were generated. Male fARKO mice on normal diet had reduced perigonadal fat but were hyperinsulinemic and by age 12 months, were insulin deficient in the absence of obesity. On high-fat diet, fARKO mice had impaired compensatory insulin secretion and hyperglycemia, with increased susceptibility to visceral obesity. Adipokine screening in fARKO mice revealed a selective increase in plasma and intra-adipose retinol binding protein 4 (RBP4) that preceded obesity. AR activation in murine 3T3 adipocytes downregulated RBP4 mRNA. We conclude that AR signaling in adipocytes not only protects against high-fat diet–induced visceral obesity but also regulates insulin action and glucose homeostasis, independently of adiposity. Androgen deficiency in adipocytes in mice resembles human type 2 diabetes, with early insulin resistance and evolving insulin deficiency. PMID:22415878

  4. Phenotypic Characterization of miR-92a−/− Mice Reveals an Important Function of miR-92a in Skeletal Development

    PubMed Central

    Penzkofer, Daniela; Bonauer, Angelika; Fischer, Ariane; Tups, Alexander; Brandes, Ralf P.; Zeiher, Andreas M.; Dimmeler, Stefanie

    2014-01-01

    MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17∼92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17∼92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17∼92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a−/− mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a−/− mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a−/− mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect. PMID:24979655

  5. Phenotypic characterization of miR-92a-/- mice reveals an important function of miR-92a in skeletal development.

    PubMed

    Penzkofer, Daniela; Bonauer, Angelika; Fischer, Ariane; Tups, Alexander; Brandes, Ralf P; Zeiher, Andreas M; Dimmeler, Stefanie

    2014-01-01

    MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17∼92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17∼92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17∼92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a-/- mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a-/- mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a-/- mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect.

  6. 6-Gingerdiols as the Major Metabolites of 6-Gingerol in Cancer Cells and in Mice and Their Cytotoxic Effects on Human Cancer Cells

    PubMed Central

    Lv, Lishuang; Chen, Huadong; Soroka, Dominique; Chen, Xiaoxin; Leung, TinChung; Sang, Shengmin

    2012-01-01

    6-Gingerol, a major pungent component of ginger (Zingiber officinale Roscoe, Zingiberaceae), has been reported to have anti-tumor activities. However, the metabolic fate of 6-gingerol and the contribution of its metabolites to the observed activities are still unclear. In the present study, we investigated the biotransformation of 6-gingerol in different cancer cells and in mice, purified and identified the major metabolites from human lung cancer cells, and determined the effects of the major metabolites on the proliferation of human cancer cells. Our results show that 6-gingerol is extensively metabolized in H-1299 human lung cancer cells, CL-13 mouse lung cancer cells, HCT-116 and HT-29 human colon cancer cells, and in mice. The two major metabolites in H-1299 cells were purified and identified as (3R,5S)-6-gingerdiol (M1) and (3S,5S)-6-gingerdiol (M2) based on the analysis of their 1D and 2D NMR data. Both metabolites induced cytotoxicity in cancer cells after 24 hours, with M1 having a comparable effect to 6-gingerol in H-1299 cells. PMID:23066935

  7. 2D-DIGE analysis of mango (Mangifera indica L.) fruit reveals major proteomic changes associated with ripening.

    PubMed

    Andrade, Jonathan de Magalhães; Toledo, Tatiana Torres; Nogueira, Silvia Beserra; Cordenunsi, Beatriz Rosana; Lajolo, Franco Maria; do Nascimento, João Roberto Oliveira

    2012-06-18

    A comparative proteomic investigation between the pre-climacteric and climacteric mango fruits (cv. Keitt) was performed to identify protein species with variable abundance during ripening. Proteins were phenol-extracted from fruits, cyanine-dye-labeled, and separated on 2D gels at pH 4-7. Total spot count of about 373 proteins spots was detected in each gel and forty-seven were consistently different between pre-climacteric and climacteric fruits and were subjected to LC-MS/MS analysis. Functional classification revealed that protein species involved in carbon fixation and hormone biosynthesis decreased during ripening, whereas those related to catabolism and the stress-response, including oxidative stress and abiotic and pathogen defense factors, accumulated. In relation to fruit quality, protein species putatively involved in color development and pulp softening were also identified. This study on mango proteomics provides an overview of the biological processes that occur during ripening.

  8. Metaproteomics reveals major microbial players and their biodegradation functions in a large-scale aerobic composting plant

    PubMed Central

    Liu, Dongming; Li, Mingxiao; Xi, Beidou; Zhao, Yue; Wei, Zimin; Song, Caihong; Zhu, Chaowei

    2015-01-01

    Composting is an appropriate management alternative for municipal solid waste; however, our knowledge about the microbial regulation of this process is still scare. We employed metaproteomics to elucidate the main biodegradation pathways in municipal solid waste composting system across the main phases in a large-scale composting plant. The investigation of microbial succession revealed that Bacillales, Actinobacteria and Saccharomyces increased significantly with respect to abundance in composting process. The key microbiologic population for cellulose degradation in different composting stages was different. Fungi were found to be the main producers of cellulase in earlier phase. However, the cellulolytic fungal communities were gradually replaced by a purely bacterial one in active phase, which did not support the concept that the thermophilic fungi are active through the thermophilic phase. The effective decomposition of cellulose required the synergy between bacteria and fungi in the curing phase. PMID:25989417

  9. Metaproteomics reveals major microbial players and their biodegradation functions in a large-scale aerobic composting plant.

    PubMed

    Liu, Dongming; Li, Mingxiao; Xi, Beidou; Zhao, Yue; Wei, Zimin; Song, Caihong; Zhu, Chaowei

    2015-11-01

    Composting is an appropriate management alternative for municipal solid waste; however, our knowledge about the microbial regulation of this process is still scare. We employed metaproteomics to elucidate the main biodegradation pathways in municipal solid waste composting system across the main phases in a large-scale composting plant. The investigation of microbial succession revealed that Bacillales, Actinobacteria and Saccharomyces increased significantly with respect to abundance in composting process. The key microbiologic population for cellulose degradation in different composting stages was different. Fungi were found to be the main producers of cellulase in earlier phase. However, the cellulolytic fungal communities were gradually replaced by a purely bacterial one in active phase, which did not support the concept that the thermophilic fungi are active through the thermophilic phase. The effective decomposition of cellulose required the synergy between bacteria and fungi in the curing phase.

  10. Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.

    PubMed

    Rissanen, Ilona; Grimes, Jonathan M; Pawlowski, Alice; Mäntynen, Sari; Harlos, Karl; Bamford, Jaana K H; Stuart, David I

    2013-05-07

    It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.

  11. Cultivar-Based Introgression Mapping Reveals Wild Species-Derived Pm-0, the Major Powdery Mildew Resistance Locus in Squash

    PubMed Central

    Holdsworth, William L.; LaPlant, Kyle E.; Bell, Duane C.; Jahn, Molly M.; Mazourek, Michael

    2016-01-01

    Powdery mildew is a major fungal disease on squash and pumpkin (Cucurbita spp.) in the US and throughout the world. Genetic resistance to the disease is not known to occur naturally within Cucurbita pepo and only infrequently in Cucurbita moschata, but has been achieved in both species through the introgression of a major resistance gene from the wild species Cucurbita okeechobeensis subsp. martinezii. At present, this gene, Pm-0, is used extensively in breeding, and is found in nearly all powdery mildew-resistant C. pepo and C. moschata commercial cultivars. In this study, we mapped C. okeechobeensis subsp. martinezii-derived single nucleotide polymorphism (SNP) alleles in a set of taxonomically and morphologically diverse and resistant C. pepo and C. moschata cultivars bred at Cornell University that, by common possession of Pm-0, form a shared-trait introgression panel. High marker density was achieved using genotyping-by-sequencing, which yielded over 50,000 de novo SNP markers in each of the three Cucurbita species genotyped. A single 516.4 kb wild-derived introgression was present in all of the resistant cultivars and absent in a diverse set of heirlooms that predated the Pm-0 introgression. The contribution of this interval to powdery mildew resistance was confirmed by association mapping in a C. pepo cultivar panel that included the Cornell lines, heirlooms, and 68 additional C. pepo cultivars and with an independent F2 population derived from C. okeechobeensis subsp. martinezii x C. moschata. The interval was refined to a final candidate interval of 76.4 kb and CAPS markers were developed inside this interval to facilitate marker-assisted selection. PMID:27936008

  12. Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing

    PubMed Central

    Törnqvist, Elin; Annas, Anita; Granath, Britta; Jalkesten, Elisabeth; Cotgreave, Ian; Öberg, Mattias

    2014-01-01

    The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level. PMID:25054864

  13. Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing.

    PubMed

    Törnqvist, Elin; Annas, Anita; Granath, Britta; Jalkesten, Elisabeth; Cotgreave, Ian; Öberg, Mattias

    2014-01-01

    The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.

  14. Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

    PubMed

    Osanai-Futahashi, M; Tatematsu, K-I; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

    2016-02-01

    Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

  15. Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

    PubMed Central

    Osanai-Futahashi, M; Tatematsu, K-i; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

    2016-01-01

    Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects. PMID:26328757

  16. Cultivar-Based Introgression Mapping Reveals Wild Species-Derived Pm-0, the Major Powdery Mildew Resistance Locus in Squash.

    PubMed

    Holdsworth, William L; LaPlant, Kyle E; Bell, Duane C; Jahn, Molly M; Mazourek, Michael

    2016-01-01

    Powdery mildew is a major fungal disease on squash and pumpkin (Cucurbita spp.) in the US and throughout the world. Genetic resistance to the disease is not known to occur naturally within Cucurbita pepo and only infrequently in Cucurbita moschata, but has been achieved in both species through the introgression of a major resistance gene from the wild species Cucurbita okeechobeensis subsp. martinezii. At present, this gene, Pm-0, is used extensively in breeding, and is found in nearly all powdery mildew-resistant C. pepo and C. moschata commercial cultivars. In this study, we mapped C. okeechobeensis subsp. martinezii-derived single nucleotide polymorphism (SNP) alleles in a set of taxonomically and morphologically diverse and resistant C. pepo and C. moschata cultivars bred at Cornell University that, by common possession of Pm-0, form a shared-trait introgression panel. High marker density was achieved using genotyping-by-sequencing, which yielded over 50,000 de novo SNP markers in each of the three Cucurbita species genotyped. A single 516.4 kb wild-derived introgression was present in all of the resistant cultivars and absent in a diverse set of heirlooms that predated the Pm-0 introgression. The contribution of this interval to powdery mildew resistance was confirmed by association mapping in a C. pepo cultivar panel that included the Cornell lines, heirlooms, and 68 additional C. pepo cultivars and with an independent F2 population derived from C. okeechobeensis subsp. martinezii x C. moschata. The interval was refined to a final candidate interval of 76.4 kb and CAPS markers were developed inside this interval to facilitate marker-assisted selection.

  17. Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding*

    PubMed Central

    Gajda, Angela M.; Zhou, Yin Xiu; Agellon, Luis B.; Fried, Susan K.; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-01-01

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP−/− and LFABP−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP−/− mice, whereas LFABP−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP−/− mice fed high fat diets became obese relative to WT, whereas IFABP−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP−/− mice preferentially utilizing lipids, and IFABP−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP−/− mice, result in divergent downstream effects at the systemic level. PMID:23990461

  18. Differential effects of prenatal and postnatal expressions of mutant human DISC1 on neurobehavioral phenotypes in transgenic mice: evidence for neurodevelopmental origin of major psychiatric disorders.

    PubMed

    Ayhan, Y; Abazyan, B; Nomura, J; Kim, R; Ladenheim, B; Krasnova, I N; Sawa, A; Margolis, R L; Cadet, J L; Mori, S; Vogel, M W; Ross, C A; Pletnikov, M V

    2011-03-01

    Strong genetic evidence implicates mutations and polymorphisms in the gene Disrupted-In-Schizophrenia-1 (DISC1) as risk factors for both schizophrenia and mood disorders. Recent studies have shown that DISC1 has important functions in both brain development and adult brain function. We have described earlier a transgenic mouse model of inducible expression of mutant human DISC1 (hDISC1) that acts in a dominant-negative manner to induce the marked neurobehavioral abnormalities. To gain insight into the roles of DISC1 at various stages of neurodevelopment, we examined the effects of mutant hDISC1 expressed during (1) only prenatal period, (2) only postnatal period, or (3) both periods. All periods of expression similarly led to decreased levels of cortical dopamine (DA) and fewer parvalbumin-positive neurons in the cortex. Combined prenatal and postnatal expression produced increased aggression and enhanced response to psychostimulants in male mice along with increased linear density of dendritic spines on neurons of the dentate gyrus of the hippocampus, and lower levels of endogenous DISC1 and LIS1. Prenatal expression only resulted in smaller brain volume, whereas selective postnatal expression gave rise to decreased social behavior in male mice and depression-like responses in female mice as well as enlarged lateral ventricles and decreased DA content in the hippocampus of female mice, and decreased level of endogenous DISC1. Our data show that mutant hDISC1 exerts differential effects on neurobehavioral phenotypes, depending on the stage of development at which the protein is expressed. The multiple and diverse abnormalities detected in mutant DISC1 mice are reminiscent of findings in major mental diseases.

  19. Identification of immunodominant Leishmania major antigenic markers of the early C57BL/6 and BALB/c mice infection stages.

    PubMed

    Sassi, Atfa; Kaak, Olfa; Elgaaied, Amel Benammar

    2015-08-24

    The C57BL/6 mouse strain is resistant to Leishmania (L.) major infection and, unlike susceptible BALB/c, develops small self healing cutaneous lesions. The specific antibody responses of C57BL/6 and BALB/c mice were previously characterized by the predominance of IgG2a ("resistant" isotype associated with Th1) and IgG1 ("pathogenic" isotype associated with Th2) antibodies, respectively. In this study, we looked for the presence of antigens able to elicit an exclusive or predominant IgG1 production during the early stages of C57BL/6 lesion development and checked whether they are recognized or not by BALB/c mice. We demonstrate first that IgG2a predominance in C57BL/6 sera occurs only late after infection whereas in BALB/c, IgG1 antibodies dominate mostly in the early stages. Interestingly, soon after inoculation of live amastigotes, C57BL/6 displayed an exclusive IgG1 reactivity against particular L. major antigens but with MWs different from those identified in BALB/c. Furthermore, mice immunized with killed amastigotes displayed striking differences in their immunodetection profiles, particularly for the IgG1 isotype. Taken together, the observed differences in the specific antibody repertoires between infected mice resulted, at least in part, from immunological events independent from those triggered by the replicating parasite, and bring new insights into the selection of future vaccine candidates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice.

    PubMed

    Iborra, Salvador; Soto, Manuel; Carrión, Javier; Nieto, Ana; Fernández, Edgar; Alonso, Carlos; Requena, Jose M

    2003-11-01

    In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-gamma) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4(+) and CD8(+) T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-gamma. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

  1. Behavioural changes are a major contributing factor in the reduction of sarcopenia in caloric-restricted ageing mice

    PubMed Central

    van Norren, Klaske; Rusli, Fenni; van Dijk, Miriam; Lute, Carolien; Nagel, Jolanda; Dijk, Francina J; Dwarkasing, Jvalini; Boekschoten, Mark V; Luiking, Yvette; Witkamp, Renger F; Müller, Michael; Steegenga, Wilma T

    2015-01-01

    Background In rodent models, caloric restriction (CR) with maintenance of adequate micronutrient supply has been reported to increase lifespan and to reduce age-induced muscle loss (sarcopenia) during ageing. In the present study, we further investigated effects of CR on the onset and severity of sarcopenia in ageing male C57BL/6 J mice. The aim of this study was to investigate whether CR induces changes in behaviour of the animals that could contribute to the pronounced health-promoting effects of CR in rodents. In addition, we aimed to investigate in more detail the effects of CR on the onset and severity of sarcopenia. Methods The mice received either an ad libitum diet (control) or a diet matching 70 E% of the control diet (C). Daily activity, body composition (dual energy X-ray absorptiometry), grip strength, insulin sensitivity, and general agility and balance were determined at different ages. Mice were killed at 4, 12, 24, and 28 months. Skeletal muscles of the hind limb were dissected, and the muscle extensor digitorum longus muscle was used for force-frequency measurements. The musculus tibialis was used for real-time quantitative PCR analysis. Results From the age of 12 months, CR animals were nearly half the weight of the control animals, which was mainly related to a lower fat mass. In the control group, the hind limb muscles showed a decline in mass at 24 or 28 months of age, which was not present in the CR group. Moreover, insulin sensitivity (oral glucose tolerance test) was higher in this group and the in vivo and ex vivo grip strength did not differ between the two groups. In the hours before food was provided, CR animals were far more active than control animals, while total daily activity was not increased. Moreover, agility test indicated that CR animals were better climbers and showed more climbing behaviours. Conclusions Our study confirms earlier findings that in CR animals less sarcopenia is present. The mice on the CR diet, however, showed

  2. Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

    SciTech Connect

    Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

    2012-10-24

    High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

  3. Single-Cell RNAseq Reveals That Pancreatic β-Cells From Very Old Male Mice Have a Young Gene Signature.

    PubMed

    Xin, Yurong; Okamoto, Haruka; Kim, Jinrang; Ni, Min; Adler, Christina; Cavino, Katie; Na, Erqian; Murphy, Andrew J; Yancopoulos, George D; Lin, Calvin; Gromada, Jesper

    2016-09-01

    Aging improves pancreatic β-cell function in mice. This is a surprising finding because aging is typically associated with functional decline. We performed single-cell RNA sequencing of β-cells from 3- and 26-month-old mice to explore how changes in gene expression contribute to improved function with age. The old mice were healthy and had reduced blood glucose levels and increased β-cell mass, which correlated to their body weight. β-Cells from young and old mice had similar transcriptome profiles. In fact, only 193 genes (0.89% of all detected genes) were significantly regulated (≥2-fold; false discovery rate < 0.01; normalized counts > 5). Of these, 183 were down-regulated and mainly associated with pathways regulating gene expression, cell cycle, cell death, and survival as well as cellular movement, function, and maintenance. Collectively our data show that β-cells from very old mice have transcriptome profiles similar to those of young mice. These data support previous findings that aging is not associated with reduced β-cell mass or functional β-cell decline in mice.

  4. Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors.

    PubMed

    Ye, Fan; Breslow, David K; Koslover, Elena F; Spakowitz, Andrew J; Nelson, W James; Nachury, Maxence V

    2013-08-06

    The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined. DOI:http://dx.doi.org/10.7554/eLife.00654.001.

  5. Rise of dinosaurs reveals major body-size transitions are driven by passive processes of trait evolution

    PubMed Central

    Sookias, Roland B.; Butler, Richard J.; Benson, Roger B. J.

    2012-01-01

    A major macroevolutionary question concerns how long-term patterns of body-size evolution are underpinned by smaller scale processes along lineages. One outstanding long-term transition is the replacement of basal therapsids (stem-group mammals) by archosauromorphs, including dinosaurs, as the dominant large-bodied terrestrial fauna during the Triassic (approx. 252–201 million years ago). This landmark event preceded more than 150 million years of archosauromorph dominance. We analyse a new body-size dataset of more than 400 therapsid and archosauromorph species spanning the Late Permian–Middle Jurassic. Maximum-likelihood analyses indicate that Cope's rule (an active within-lineage trend of body-size increase) is extremely rare, despite conspicuous patterns of body-size turnover, and contrary to proposals that Cope's rule is central to vertebrate evolution. Instead, passive processes predominate in taxonomically and ecomorphologically more inclusive clades, with stasis common in less inclusive clades. Body-size limits are clade-dependent, suggesting intrinsic, biological factors are more important than the external environment. This clade-dependence is exemplified by maximum size of Middle–early Late Triassic archosauromorph predators exceeding that of contemporary herbivores, breaking a widely-accepted ‘rule’ that herbivore maximum size greatly exceeds carnivore maximum size. Archosauromorph and dinosaur dominance occurred via opportunistic replacement of therapsids following extinction, but were facilitated by higher archosauromorph growth rates. PMID:22298850

  6. Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

    PubMed

    O'Neill, Eric M; Schwartz, Rachel; Bullock, C Thomas; Williams, Joshua S; Shaffer, H Bradley; Aguilar-Miguel, X; Parra-Olea, Gabriela; Weisrock, David W

    2013-01-01

    Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

  7. Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

    PubMed Central

    Nair, Asha V.; Singh, Himansha; Raturi, Sagar; Neuberger, Arthur; Tong, Zhen; Ding, Ning; Agboh, Kelvin; van Veen, Hendrik W.

    2016-01-01

    The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport. PMID:27917857

  8. Rise of dinosaurs reveals major body-size transitions are driven by passive processes of trait evolution.

    PubMed

    Sookias, Roland B; Butler, Richard J; Benson, Roger B J

    2012-06-07

    A major macroevolutionary question concerns how long-term patterns of body-size evolution are underpinned by smaller scale processes along lineages. One outstanding long-term transition is the replacement of basal therapsids (stem-group mammals) by archosauromorphs, including dinosaurs, as the dominant large-bodied terrestrial fauna during the Triassic (approx. 252-201 million years ago). This landmark event preceded more than 150 million years of archosauromorph dominance. We analyse a new body-size dataset of more than 400 therapsid and archosauromorph species spanning the Late Permian-Middle Jurassic. Maximum-likelihood analyses indicate that Cope's rule (an active within-lineage trend of body-size increase) is extremely rare, despite conspicuous patterns of body-size turnover, and contrary to proposals that Cope's rule is central to vertebrate evolution. Instead, passive processes predominate in taxonomically and ecomorphologically more inclusive clades, with stasis common in less inclusive clades. Body-size limits are clade-dependent, suggesting intrinsic, biological factors are more important than the external environment. This clade-dependence is exemplified by maximum size of Middle-early Late Triassic archosauromorph predators exceeding that of contemporary herbivores, breaking a widely-accepted 'rule' that herbivore maximum size greatly exceeds carnivore maximum size. Archosauromorph and dinosaur dominance occurred via opportunistic replacement of therapsids following extinction, but were facilitated by higher archosauromorph growth rates.

  9. Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

    SciTech Connect

    Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

    1986-12-01

    A radioimmunoassay that makes use of whole Schistosomula and /sup 125/I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000.

  10. An alien divalent ion reveals a major role for Ca²⁺ buffering in controlling slow transmitter release.

    PubMed

    Babai, Norbert; Kochubey, Olexiy; Keller, Daniel; Schneggenburger, Ralf

    2014-09-17

    Ca(2+)-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca(2+) buffers and Ca(2+) sensors in shaping release kinetics are still controversial. Replacing extracellular Ca(2+) by Sr(2+) causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr(2+) uncaging and made quantitative Sr(2+)- and Ca(2+)-imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca(2+) sensors and Ca(2+) buffers in the control of fast and slow release. We show that Sr(2+) activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr(2+) also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr(2+) was less efficient than Ca(2+) in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr(2+) than for Ca(2+) (~5-fold). This, together with an enhanced Sr(2+) permeation through presynaptic Ca(2+) channels (~2-fold), admits a drastically higher spatially averaged Sr(2+) transient compared with Ca(2+). Together, despite the lower affinity of Sr(2+) at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr(2+) transients explain the enhanced late release. This also allows us to conclude that Ca(2+) buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca(2+). Copyright © 2014 the authors 0270-6474/14/3412622-14$15.00/0.

  11. mtDNA variation predicts population size in humans and reveals a major Southern Asian chapter in human prehistory.

    PubMed

    Atkinson, Quentin D; Gray, Russell D; Drummond, Alexei J

    2008-02-01

    The relative timing and size of regional human population growth following our expansion from Africa remain unknown. Human mitochondrial DNA (mtDNA) diversity carries a legacy of our population history. Given a set of sequences, we can use coalescent theory to estimate past population size through time and draw inferences about human population history. However, recent work has challenged the validity of using mtDNA diversity to infer species population sizes. Here we use Bayesian coalescent inference methods, together with a global data set of 357 human mtDNA coding-region sequences, to infer human population sizes through time across 8 major geographic regions. Our estimates of relative population sizes show remarkable concordance with the contemporary regional distribution of humans across Africa, Eurasia, and the Americas, indicating that mtDNA diversity is a good predictor of population size in humans. Plots of population size through time show slow growth in sub-Saharan Africa beginning 143-193 kya, followed by a rapid expansion into Eurasia after the emergence of the first non-African mtDNA lineages 50-70 kya. Outside Africa, the earliest and fastest growth is inferred in Southern Asia approximately 52 kya, followed by a succession of growth phases in Northern and Central Asia (approximately 49 kya), Australia (approximately 48 kya), Europe (approximately 42 kya), the Middle East and North Africa (approximately 40 kya), New Guinea (approximately 39 kya), the Americas (approximately 18 kya), and a second expansion in Europe (approximately 10-15 kya). Comparisons of relative regional population sizes through time suggest that between approximately 45 and 20 kya most of humanity lived in Southern Asia. These findings not only support the use of mtDNA data for estimating human population size but also provide a unique picture of human prehistory and demonstrate the importance of Southern Asia to our recent evolutionary past.

  12. Tissue-specific signaling networks rewired by major somatic mutations in human cancer revealed by proteome-wide discovery.

    PubMed

    Zhao, Junfei; Cheng, Feixiong; Zhao, Zhongming

    2017-03-31

    Massive somatic mutations discovered by large cancer genome sequencing projects provide unprecedented opportunities in the development of precision oncology. However, deep understanding of functional consequences of somatic mutations and identifying actionable mutations and the related drug responses currently remain formidable challenges. Dysfunction of protein post-translational modification plays critical roles in tumorigenesis and drug responses. In this study, we proposed a novel computational oncoproteomics approach, named kinome-wide network module for cancer pharmacogenomics (KNMPx), for identifying actionable mutations that rewired signaling networks and further characterized tumorigenesis and anticancer drug responses. Specifically, we integrated 746,631 missense mutations in 4,997 tumor samples across 16 major cancer types/subtypes from The Cancer Genome Atlas into over 170,000 carefully curated non-redundant phosphorylation sites covering 18,610 proteins. We found 47 mutated proteins (e.g., ERBB2, TP53, and CTNNB1) that had enriched missense mutations at their phosphorylation sites in pan-cancer analysis. In addition, tissue-specific kinase-substrate interaction modules altered by somatic mutations identified by KNMPx were significantly associated with patient survival. We further reported a kinome-wide landscape of pharmacogenomic interactions by incorporating somatic mutation-rewired signaling networks in 1,001 cancer cell lines via KNMPx. Interestingly, we found that cell lines could highly reproduce oncogenic phosphorylation site mutations identified in primary tumors, supporting the confidence in their associations with sensitivity/resistance of inhibitors targeting EGF, MAPK, PI3K, mTOR, and Wnt signaling pathways. In summary, this systematic oncoproteomics analysis of kinome phosphorylation site mutations illustrates new capabilities to speed the development of precision oncology.

  13. Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool*

    PubMed Central

    Olshina, Maya A.; Angley, Lauren M.; Ramdzan, Yasmin M.; Tang, Jinwei; Bailey, Michael F.; Hill, Andrew F.; Hatters, Danny M.

    2010-01-01

    Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100–6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s20,w = 140 S) and IBs (mode s20,w = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates. PMID:20444706

  14. Expression Profiling of Major Histocompatibility and Natural Killer Complex Genes Reveals Candidates for Controlling Risk of Graft versus Host Disease

    PubMed Central

    Novota, Peter; Zinöcker, Severin; Norden, Jean; Wang, Xiao Nong; Sviland, Lisbet; Opitz, Lennart; Salinas-Riester, Gabriela; Dressel, Ralf

    2011-01-01

    Background The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. Methodology/Principal Findings To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. Conclusions/Significance We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients. PMID:21305040

  15. Expression profiling of major histocompatibility and natural killer complex genes reveals candidates for controlling risk of graft versus host disease.

    PubMed

    Novota, Peter; Zinöcker, Severin; Norden, Jean; Wang, Xiao Nong; Sviland, Lisbet; Opitz, Lennart; Salinas-Riester, Gabriela; Rolstad, Bent; Dickinson, Anne M; Walter, Lutz; Dressel, Ralf

    2011-01-28

    The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

  16. Anti-Leishmanial Activity (In Vitro and In Vivo) of Allicin and Allicin Cream Using Leishmania major (Sub-strain Zymowme LON4) and Balb/c Mice

    PubMed Central

    Metwally, Dina M.; Al-Olayan, Ebtesam M.; El-Khadragy, Manal F.; Alkathiri, Badriah

    2016-01-01

    Background Leishmania is a unicellular protozoan parasite that produces several human diseases, ranging from localized self-healing cutaneous lesions to deadly visceral infections. Objective The effect of allicin on the growth of Leishmania major (L. major) promastigotes was evaluated under in vitro conditions. Moreover, the efficacy of a topical allicin cream was examined in BALB/c (Bagg albino, laboratory-bred strain of the House Mouse) mice with cutaneous leishmanial lesions compared to the currently used drug, sodiumstibogluconate (pentostam). Methods Cytotoxiciy and promastigote proliferation were measured. Different concentrations (50, 100, 150, and 200 μM) of liquid allicin were tested on L. major promastigotes twice: after 24 and 48 hours using an MTT colorimetric assay. In the in vivo condition, the efficacies of allicin cream and liquid allicin at two concentrations (0.15 μM/mouse and 0.30 μM/mouse) were evaluated. Serum factors of the control and treated groups were tested to evaluate the toxic effects of allicin on the liver and kidney. Results Allicin at a concentration of 50 μM inhibited the growth of Leishmania promastigotes. Topical application of allicin cream reduced lesion sizes in mice. No significant differences in biochemical analysis were observed between the control and treated groups. Conclusions Allicin has antileishmanial effects under in vitro and in vivo conditions and may be used in clinical applications. PMID:27537199

  17. Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks

    PubMed Central

    Saini, Camille; Liani, André; Curie, Thomas; Gos, Pascal; Kreppel, Florian; Emmenegger, Yann; Bonacina, Luigi; Wolf, Jean-Pierre; Poget, Yves-Alain; Franken, Paul; Schibler, Ueli

    2013-01-01

    The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase. PMID:23824542

  18. Neutralization of both IL-1α/IL-1β plays a major role in suppressing combined cigarette smoke/virus-induced pulmonary inflammation in mice.

    PubMed

    Bucher, Hannes; Mang, Samuel; Keck, Martina; Przibilla, Michèl; Lamb, David; Schiele, Felix; Wittenbrink, Mareike; Fuchs, Klaus; Jung, Birgit; Erb, Klaus J; Peter, Daniel

    2017-03-15

    Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1β are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1β did not provide significant activity. In line with the in vivo findings, IL-1α Abs but not IL-1β Abs reduced levels of TNF-α and IL-6 in H1N1 infected primary human bronchial epithelial air-liquid-interface cell culture. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1β Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1β. Our results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to individual neutralization IL-1α or IL-1β or inhibition of the IL-1R1.

  19. Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome

    PubMed Central

    Joyce, Cailin E.; Zhou, Xiang; Xia, Jing; Ryan, Caitriona; Thrash, Breck; Menter, Alan; Zhang, Weixiong; Bowcock, Anne M.

    2011-01-01

    Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 108 small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2–42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin. PMID:21807764

  20. Targeted disruption of TGFBI in mice reveals its role in regulating bone mass and bone size through periosteal bone formation.

    PubMed

    Yu, Hongrun; Wergedal, Jon E; Zhao, Yongliang; Mohan, Subburaman

    2012-07-01

    Transforming growth factor-beta induced (TGFBI) and periostin are two closely related proteins in structure as well as in function. A previous study found that periostin positively regulates bone size. Here, we hypothesize that TGFBI has a similar function in bone development. To test this hypothesis, we employed TGFBI-deficient mice, which were generated by targeted disruption of the TGFBI gene. We bred these mice with C57BL/6J mice to generate homozygous TGFBI-deficient (TGFBI(-/-)) mice and homozygous wild-type littermates. All mice were raised to 12 weeks of age. Bone mass parameters were determined by PIXImus and micro-CT, bone strength parameters by three-point bending, and bone formation and resorption parameters by histomorphometry. We found that targeted disruption of TGFBI led to reduced body size, bone mass, bone size, and bone strength. This indicates that, like periostin, TGFBI also positively regulates bone size and that changes in bone size affect bone strength. Furthermore, there was also a significant decrease in periosteal, but not endosteal, bone formation rate of cortical bone in TGFBI(-/-) mice, suggesting that the observed effect of TGFBI on bone mass and bone size was largely caused by the effect of TGFBI on periosteal bone formation.

  1. Divergence patterns of genic copy number variation in natural populations of the house mouse (Mus musculus domesticus) reveal three conserved genes with major population-specific expansions.

    PubMed

    Pezer, Željka; Harr, Bettina; Teschke, Meike; Babiker, Hiba; Tautz, Diethard

    2015-08-01

    Copy number variation represents a major source of genetic divergence, yet the evolutionary dynamics of genic copy number variation in natural populations during differentiation and adaptation remain unclear. We applied a read depth approach to genome resequencing data to detect copy number variants (CNVs) ≥1 kb in wild-caught mice belonging to four populations of Mus musculus domesticus. We complemented the bioinformatics analyses with experimental validation using droplet digital PCR. The specific focus of our analysis is CNVs that include complete genes, as these CNVs could be expected to contribute most directly to evolutionary divergence. In total, 1863 transcription units appear to be completely encompassed within CNVs in at least one individual when compared to the reference assembly. Further, 179 of these CNVs show population-specific copy number differences, and 325 are subject to complete deletion in multiple individuals. Among the most copy-number variable genes are three highly conserved genes that encode the splicing factor CWC22, the spindle protein SFI1, and the Holliday junction recognition protein HJURP. These genes exhibit population-specific expansion patterns that suggest involvement in local adaptations. We found that genes that overlap with large segmental duplications are generally more copy-number variable. These genes encode proteins that are relevant for environmental and behavioral interactions, such as vomeronasal and olfactory receptors, as well as major urinary proteins and several proteins of unknown function. The overall analysis shows that genic CNVs contribute more to population differentiation in mice than in humans and may promote and speed up population divergence.

  2. Divergence patterns of genic copy number variation in natural populations of the house mouse (Mus musculus domesticus) reveal three conserved genes with major population-specific expansions

    PubMed Central

    Pezer, Željka; Harr, Bettina; Teschke, Meike; Babiker, Hiba; Tautz, Diethard

    2015-01-01

    Copy number variation represents a major source of genetic divergence, yet the evolutionary dynamics of genic copy number variation in natural populations during differentiation and adaptation remain unclear. We applied a read depth approach to genome resequencing data to detect copy number variants (CNVs) ≥1 kb in wild-caught mice belonging to four populations of Mus musculus domesticus. We complemented the bioinformatics analyses with experimental validation using droplet digital PCR. The specific focus of our analysis is CNVs that include complete genes, as these CNVs could be expected to contribute most directly to evolutionary divergence. In total, 1863 transcription units appear to be completely encompassed within CNVs in at least one individual when compared to the reference assembly. Further, 179 of these CNVs show population-specific copy number differences, and 325 are subject to complete deletion in multiple individuals. Among the most copy-number variable genes are three highly conserved genes that encode the splicing factor CWC22, the spindle protein SFI1, and the Holliday junction recognition protein HJURP. These genes exhibit population-specific expansion patterns that suggest involvement in local adaptations. We found that genes that overlap with large segmental duplications are generally more copy-number variable. These genes encode proteins that are relevant for environmental and behavioral interactions, such as vomeronasal and olfactory receptors, as well as major urinary proteins and several proteins of unknown function. The overall analysis shows that genic CNVs contribute more to population differentiation in mice than in humans and may promote and speed up population divergence. PMID:26149421

  3. In vivo imaging reveals selective PPAR activity in the skin of peroxisome proliferator-activated receptor responsive element-luciferase reporter mice.

    PubMed

    El-Jamal, Noura; Dubuquoy, Laurent; Auwerx, Johan; Bertin, Benjamin; Desreumaux, Pierre

    2013-02-01

    Peroxisome proliferator-activated receptors (PPARs) have been revealed as key regulators of several skin disorders. This has led to a growing interest in the development of drugs targeting PPARs as therapeutics for skin diseases. To evaluate skin PPAR activity, we developed peroxisome proliferator responsive element-luciferase (PPRE-Luc) mice, a mouse model in which the luciferase gene expression is under the control of a PPAR-inducible promoter in all organs. Our aim was to define and validate experimental conditions to establish PPRE-Luc mice as a valuable tool for in vivo non-invasive evaluation of PPARs activation in the skin. We demonstrated by optical imaging that topical application of 40 mm of Luciferin for 10 min was enough to reveal the optimal luciferase activity in mice skin. The treatment of mice skin with the PPARγ and PPARα agonists, pioglitazone and WY14643, was associated with significant increase in photons emission reaching maximal signalling at 6 h. We have performed dose response studies by testing a large range of pioglitazone and WY14643 concentrations on mouse skin. The specificity of bioluminescence signal induced by pioglitazone and WY14643 was assessed using PPARγ and PPARα antagonists, GW9662 and GW6471, respectively. This approach revealed that the isoform specificity of PPARs agonists decreased when high ligand concentrations were applied on mouse skin. These results were further confirmed by in vitro measurement of luciferase activity in skin extracts. Overall, our results demonstrated that PPRE-Luc mice represent a valuable reporter mouse model for the in vivo pharmacological profiling of drugs targeting PPARs in the skin. © 2012 John Wiley & Sons A/S.

  4. Roles of the 15-kDa selenoprotein (Sep15) in redox homeostasis and cataract development revealed by the analysis of Sep 15 knockout mice.

    PubMed

    Kasaikina, Marina V; Fomenko, Dmitri E; Labunskyy, Vyacheslav M; Lachke, Salil A; Qiu, Wenya; Moncaster, Juliet A; Zhang, Jie; Wojnarowicz, Mark W; Natarajan, Sathish Kumar; Malinouski, Mikalai; Schweizer, Ulrich; Tsuji, Petra A; Carlson, Bradley A; Maas, Richard L; Lou, Marjorie F; Goldstein, Lee E; Hatfield, Dolph L; Gladyshev, Vadim N

    2011-09-23

    The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.

  5. Cyclic Alopecia and Abnormal Epidermal Cornification in Zdhhc13-Deficient Mice Reveal the Importance of Palmitoylation in Hair and Skin Differentiation.

    PubMed

    Liu, Kai-Ming; Chen, Yi-Ju; Shen, Li-Fen; Haddad, Amir N S; Song, I-Wen; Chen, Li-Ying; Chen, Yu-Ju; Wu, Jer-Yuarn; Yen, Jeffrey J Y; Chen, Yuan-Tsong

    2015-11-01

    Many biochemical pathways involved in hair and skin development have not been investigated. Here, we reported on the lesions and investigated the mechanism underlying hair and skin abnormalities in Zdhhc13(skc4) mice with a deficiency in DHHC13, a palmitoyl-acyl transferase encoded by Zdhhc13. Homozygous affected mice showed ragged and dilapidated cuticle of the hair shaft (CUH, a hair anchoring structure), poor hair anchoring ability, and premature hair loss at early telogen phase of the hair cycle, resulting in cyclic alopecia. Furthermore, the homozygous affected mice exhibited hyperproliferation of the epidermis, disturbed cornification, fragile cornified envelope (CE, a skin barrier structure), and impaired skin barrier function. Biochemical investigations revealed that cornifelin, which contains five palmitoylation sites at cysteine residues (C58, C59, C60, C95, and C101), was a specific substrate of DHHC13 and that it was absent in the CUH and CE structures of the affected mice. Furthermore, cornifelin levels were markedly reduced when two palmitoylated cysteines were replaced with serine (C95S and C101S). Taken together, our results suggest that DHHC13 is important for hair anchoring and skin barrier function and that cornifelin deficiency contributes to cyclic alopecia and skin abnormalities in Zdhhc13(skc4) mice.

  6. A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

    SciTech Connect

    Bender, M.A. . Dept. of Pathology); Gelinas, R.E.; Miller )

    1989-04-01

    Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

  7. Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

    PubMed Central

    Souza, A R; Gesztesi, J -L; Moraes, J Z; Cruz, C R B; Sato, J; Mariano, M; Lopes, J D

    1998-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network. PMID:9764601

  8. The Beneficial Effects of Combined Grape Pomace and Omija Fruit Extracts on Hyperglycemia, Adiposity and Hepatic Steatosis in db/db Mice: A Comparison with Major Index Compounds

    PubMed Central

    Cho, Su-Jung; Park, Hae-Jin; Jung, Un Ju; Kim, Hye-Jin; Moon, Byoung Seok; Choi, Myung-Sook

    2014-01-01

    This study investigated the effects of combined grape pomace and omija fruit extracts (GO) on diabetes-related metabolic changes in type 2 diabetic db/db mice. The effects of GO were compared with those of a resveratrol and schizandrin mixture (RS), which is a mixture of major components of GO. Mice were fed a normal diet with RS (0.005% resveratrol and 0.02% schizandrin in diet, w/w) or GO (0.3% grape pomace ethanol extract and 0.05% omija fruit ethanol extract in diet, w/w) for seven weeks. RS and GO not only lowered the levels of blood and plasma glucose, HbA1c, insulin and homeostasis model assessment of insulin resistance (HOMA-IR) with a simultaneous decrease in hepatic gluconeogenic enzymes activities and adiposity, but also improved preservation of the pancreatic β-cells. Plasma leptin and resistin levels were lower while the plasma adiponectin level was higher in the RS and GO groups than in the control group. Especially, GO increased hepatic glucokinase activity and gene expression and improved hepatic steatosis by elevating fatty acid oxidation compared to RS. These findings suggest that GO ameliorates hyperglycemia, adiposity and hepatic steatosis in type 2 diabetic mice. PMID:25272231

  9. Peganum harmala Aqueous and Ethanol Extracts Effects on Lesions Caused by Leishmania major (MRHO/IR/75/ER) in BALB/c Mice

    PubMed Central

    Khoshzaban, Fariba; Ghaffarifar, Fatemeh; Jamshidi Koohsari, Hamid Reza

    2014-01-01

    Background: Leishmaniasis is one of the six most common parasitic infections in the tropical regions. There are different therapeutic modalities, however therapeutic resistance is developed and resulted in numerous problems. Therefore, evaluation of other therapeutic modalities is performed extensively. Objectives: The current study aimed to compare the therapeutic response of cutaneous leishmaniasis with Glucantime and Peganum harmala extracts (aqueous and ethanol) in the animal model. Materials and Methods: The therapeutic response of Leishmania major to Glucantime and P. harmala extracts (aqueous and ethanol) in animal model was studied in BALB/c mice. These mice were divided into four groups according to receiving either one of these three agents, and the control group. The therapeutic response was evaluated according to the parasitic load before and after treatment and also with measuring the size of the lesions. Results: The results showed that ethanol extract of P. harmala had good therapeutic efficacy in treatment of lesions in mice (P < 0.05), and the efficacy was significant in the eighth week after the treatment. There was also a statistically significant difference between the groups regarding the parasitic load (P < 0.05). Conclusions: According to the current study results, it may be concluded that ethanol extract of P. harmala is efficient in the treatment of cutaneous leishmaniasis, and the efficiency is comparable with that of Glucantime. PMID:25368792

  10. A Strong Anti-Inflammatory Signature Revealed by Liver Transcription Profiling of Tmprss6−/− Mice

    PubMed Central

    Riba, Michela; Rausa, Marco; Sorosina, Melissa; Cittaro, Davide; Garcia Manteiga, Jose Manuel; Nai, Antonella; Pagani, Alessia; Martinelli-Boneschi, Filippo; Stupka, Elia; Camaschella, Clara; Silvestri, Laura

    2013-01-01

    Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe−/− mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe−/− deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6. PMID:23922777

  11. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regions and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.

  12. Differential immune response of congenic mice to ultraviolet-treated major histocompatibility complex class II-incompatible skin grafts

    SciTech Connect

    Vermeer, B.J.; Santerse, B.; Van De Kerckhove, B.A.; Schothorst, A.A.; Claas, F.H.

    1988-03-01

    The influence of ultraviolet (UVB) irradiation on the survival of H-2 class II-disparate skin grafts was studied in congenic mouse strains. Isolated skin was UVB irradiated in vitro at a dose of 40 mJ/cm/sup 2/ from both sides to remove Ia immunogenicity. Immediately after irradiation the skin was transplanted onto the flank of allogeneic mice. When B10.AQR grafts were transplanted onto B10.T(6R) recipients, a significant prolongation of the survival time was observed, while 50% of the UVB-treated grafts were not rejected at all. However, in the opposite direction--i.e., B10.T(6R) grafts onto B10.AQR recipients, no significant prolongation of the survival was observed. To test whether this effect was due to a difference in susceptibility of the donor skin to UVB irradiation or to a different immune response in the recipients, (B10.T(6R) x B10.AQR) grafts were transplanted onto the parent strains. Similar results were obtained, in that UVB-treated grafts did not show a prolonged survival in B10.AQR recipients, whereas a significant prolongation (50% of the grafts survived more than 100 days) was observed in B10.T(6R) recipients. UVB-treated (B10.T(6R) x B10.AQR)F1 grafts were also transplanted onto (B10.T(6R) x C57B1/10)F1, (B10.AQR x C57B1/10)F1, (B10.T(6R) x Balb/c)F1 and (B10.AQR x Balb/c)F1 recipients--but in none of these combinations was a prolonged survival time observed. These data suggest that, in contrast to all in vitro experiments, the abrogation of the immune response by UVB treatment of the stimulator cells is, in vivo, not a general phenomenon. The genetic constitution of the responder mice seems to play an important role in determining whether or not an immune response takes place.

  13. Cellular density, a major factor involved in PDT cytotoxic responses: Study on three different lines of human retinoblastoma grafted on nude mice.

    PubMed

    Thomas, Carole D; Poyer, Florent; Maillard, Philippe; Chauvin, Benoit; Lupu, Mihaela; Mispelter, Joël

    2015-06-01

    PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice. Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval. First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology. We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein L-isoaspartate O-methyltransferase-deficient mice.

    PubMed

    Dai, Shujia; Ni, Wenqin; Patananan, Alexander N; Clarke, Steven G; Karger, Barry L; Zhou, Zhaohui Sunny

    2013-02-19

    The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

  15. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

    PubMed

    Soto, Manuel; Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M; Martín, M Elena; Alonso, Carlos; Coelho, Eduardo A F; Barral, Aldina; Barral-Netto, Manoel; Iborra, Salvador

    2015-05-01

    Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

  16. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  17. Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice.

    PubMed

    Doyle, Alfred D; Jacobsen, Elizabeth A; Ochkur, Sergei I; McGarry, Michael P; Shim, Kevin G; Nguyen, David T C; Protheroe, Cheryl; Colbert, Dana; Kloeber, Jake; Neely, Joseph; Shim, Kelly P; Dyer, Kimberly D; Rosenberg, Helene F; Lee, James J; Lee, Nancy A

    2013-08-01

    Eosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

  18. Molecular Regulation of Sexual Preference Revealed by Genetic Studies of 5-HT in the Brain of Male Mice

    PubMed Central

    Liu, Yan; Jiang, Yun’ai; Si, Yunxia; Kim, Ji-Young; Chen, Zhou-Feng; Rao, Yi

    2014-01-01

    To whom should a male directs his mating? While it is a critical social interaction, little is known about molecular and cellular mechanisms controlling mammalian sexual preference. Here we report that the neurotransmitter 5-HT is required for male sexual preference. Male mice lacking central serotonergic neurons lost sexual preference but were not generally defective in olfaction. A role for 5-hydroxytryptamine (5-HT) was demonstrated by the phenotype of mice unable to synthesize 5-HT in the brain when lacking tryptophan hydroxylase 2 (Tph2). 5-hydroxytryptophan (5-HTP) injection rescued the phenotype of adult Tph2 knockout mice within 35 minutes. These results indicate that 5-HT and serotonergic neurons in the adult brain regulate mammalian sexual preference. PMID:21441904

  19. Sustained Delivery of NT-3 from Lens Fiber Cells in Transgenic Mice Reveals Specificity of Neuroprotection in Retinal Degenerations

    PubMed Central

    LaVail, Matthew M.; Nishikawa, Shimpei; Duncan, Jacque L.; Yang, Haidong; Matthes, Michael T.; Yasumura, Douglas; Vollrath, Douglas; Overbeek, Paul A.; Ash, John D.; Robinson, Michael L.

    2009-01-01

    Several neurotrophic factors (NTFs) are effective in protecting retinal photoreceptor cells from the damaging effects of constant light and slowing the rate of inherited photoreceptor degenerations. It is currently unclear whether, if continuously available, all NTFs can be protective for many or most retinal degenerations (RDs). We have used transgenic mice that continuously overexpress the neurotrophin, NT-3, from lens fibers under the control of the αA-crystallin promoter to test for neuroprotection in light-damage experiments and in four naturally occurring or transgenically induced RDs in mice. Lens-specific expression of NT-3 mRNA was demonstrated both by in situ hybridization in embryos and by RT-PCR in adult mice. Furthermore, NT-3 protein was found in abundance in the lens, ocular fluids and retina by ELISA and immunocytochemistry. Overexpression of NT-3 had no adverse effects on the structure or function of the retina for up to at least 14 months of age. Mice expressing the NT-3 transgene were protected from the damaging effects of constant light to a much greater degree than those receiving bolus injections of NT-3. When the NT-3 transgene was transferred into rd/rd, Rds/+, Q344ter mutant rhodopsin or Mertk knockout mice, overexpression of NT-3 had no protective effect on the RDs in these mice. Thus, specificity of the neuroprotective effect of NT-3 is clearly demonstrated, and different molecular mechanisms are inferred to mediate the protective effect in light-induced and inherited RDs. PMID:18925574

  20. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis.

    PubMed

    Zhang, Yong; Goldman, Scott; Baerga, Rebecca; Zhao, Yun; Komatsu, Masaaki; Jin, Shengkan

    2009-11-24

    White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, approximately 50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of beta-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect.

  1. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis

    PubMed Central

    Zhang, Yong; Goldman, Scott; Baerga, Rebecca; Zhao, Yun; Komatsu, Masaaki; Jin, Shengkan

    2009-01-01

    White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, ≈50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of β-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect. PMID:19910529

  2. Live imaging of bioluminescent leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics.

    PubMed

    Ratet, Gwenn; Veyrier, Frédéric J; Fanton d'Andon, Martine; Kammerscheit, Xavier; Nicola, Marie-Anne; Picardeau, Mathieu; Boneca, Ivo G; Werts, Catherine

    2014-12-01

    Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to

  3. Genetic Mapping in Mice Reveals the Involvement of Pcdh9 in Long-Term Social and Object Recognition and Sensorimotor Development.

    PubMed

    Bruining, Hilgo; Matsui, Asuka; Oguro-Ando, Asami; Kahn, René S; Van't Spijker, Heleen M; Akkermans, Guus; Stiedl, Oliver; van Engeland, Herman; Koopmans, Bastijn; van Lith, Hein A; Oppelaar, Hugo; Tieland, Liselotte; Nonkes, Lourens J; Yagi, Takeshi; Kaneko, Ryosuke; Burbach, J Peter H; Yamamoto, Nobuhiko; Kas, Martien J

    2015-10-01

    Quantitative genetic analysis of basic mouse behaviors is a powerful tool to identify novel genetic phenotypes contributing to neurobehavioral disorders. Here, we analyzed genetic contributions to single-trial, long-term social and nonsocial recognition and subsequently studied the functional impact of an identified candidate gene on behavioral development. Genetic mapping of single-trial social recognition was performed in chromosome substitution strains, a sophisticated tool for detecting quantitative trait loci (QTL) of complex traits. Follow-up occurred by generating and testing knockout (KO) mice of a selected QTL candidate gene. Functional characterization of these mice was performed through behavioral and neurological assessments across developmental stages and analyses of gene expression and brain morphology. Chromosome substitution strain 14 mapping studies revealed an overlapping QTL related to long-term social and object recognition harboring Pcdh9, a cell-adhesion gene previously associated with autism spectrum disorder. Specific long-term social and object recognition deficits were confirmed in homozygous (KO) Pcdh9-deficient mice, while heterozygous mice only showed long-term social recognition impairment. The recognition deficits in KO mice were not associated with alterations in perception, multi-trial discrimination learning, sociability, behavioral flexibility, or fear memory. Rather, KO mice showed additional impairments in sensorimotor development reflected by early touch-evoked biting, rotarod performance, and sensory gating deficits. This profile emerged with structural changes in deep layers of sensory cortices, where Pcdh9 is selectively expressed. This behavior-to-gene study implicates Pcdh9 in cognitive functions required for long-term social and nonsocial recognition. This role is supported by the involvement of Pcdh9 in sensory cortex development and sensorimotor phenotypes. Copyright © 2015 Society of Biological Psychiatry. Published

  4. Mesenchymal stem cells alter macrophage immune responses to Leishmania major infection in both susceptible and resistance mice.

    PubMed

    Dameshghi, Safura; Zavaran-Hosseini, Ahmad; Soudi, Sara; Shirazi, Fatemeh Jalali; Nojehdehi, Shahrzad; Hashemi, Seyed Mahmoud

    2016-02-01

    Mesenchymal stem cells (MSCs) are attracted to inflammation site and switch immune system to modulate inflammatory responses. This ability makes MSCs the best candidate cells for stem cell therapy of infection diseases. Therefore, we aimed to evaluate the modulatory effect of adipose-derived MSCs (AD-MSCs) on macrophages in Leishmania (L.) major infection. Macrophages and MSCs were isolated from both susceptible (BALB/c) and resistance (C57BL/6) strains. After co-culture of AD-MSCs with macrophages using a transwell system, we assessed MSCs-educated macrophage responses to L. major infection. Our results indicated suppression in levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) of MSCs co-cultured macrophages in response to L. major infection. To clarify the effects of this suppression on inflammatory conditions, TNF-α/IL-10 ratio was calculated, indicating an increase in TNF-α/IL-10 ratio in MSCs co-cultured groups. The higher TNF-α/IL-10 ratio was observed in BALB/c macrophages co-cultured with BALB/c MSCs. Nitric oxide (NO) assay presented a significant reduction in the supernatant of all MSCs co-cultured groups compared to control. We observed a significant reduction in phagocytosis of MSCs co-cultured groups in response to L. major infection without any significant differences in the phagocytic index. In conclusion, our results represented a new spectrum of immunomodulation induced by MSCs co-cultured with macrophages in response to L. major infection. The magnitude of immunoregulation was different between BALB/c and C57BL/6 strains. Our findings also showed that MSCs exerted potential effect of M1 polarization due to unequal decrease in levels of TNF-α and IL-10 when we considered TNF-α and IL-10as representatives of M1 and M2 phenotypes, respectively. Induction of inflammatory cytokine milieu and reduction in level of IL-10 provides a new hope for stem cell therapy of leishmaniasis in susceptible models.

  5. Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

    PubMed

    Giniatullina, A; Maroteaux, G; Geerts, C J; Koopmans, B; Loos, M; Klaassen, R; Chen, N; van der Schors, R C; van Nierop, P; Li, K W; de Jong, J; Altrock, W D; Cornelisse, L N; Toonen, R F; van der Sluis, S; Sullivan, P F; Stiedl, O; Posthuma, D; Smit, A B; Groffen, A J; Verhage, M

    2015-08-06

    Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

  6. Possible mechanisms of thyroid hormone disruption in mice by BDE 47, a major polybrominated diphenyl ether congener

    SciTech Connect

    Richardson, Vicki M. Staskal, Daniele F.; Ross, David G.; Diliberto, Janet J.; DeVito, Michael J.; Birnbaum, Linda S.

    2008-02-01

    Polybrominated diphenyl ethers (PBDEs) are a class of polyhalogenated aromatic compounds commercially used as fire retardants in consumer products. These compounds have been shown to decrease thyroid hormone concentrations in rodents after acute exposures. This study examines the ability of 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) to decrease circulating thyroid hormone concentrations and pairs this with BDE 47-induced effects on genes involved in thyroid hormone homeostasis. Female C57BL/6 mice (9 weeks old) were orally administered 3, 10, or 100 mg/kg/day of BDE 47 for 4 days. Animals were euthanized 24 h after the final dose (day 5) and liver, kidney, and serum were collected for analysis. BDE 47 caused a significant 43% decrease at 100 mg/kg/day in serum total thyroxine (T{sub 4}) concentrations. There was no increase in hepatic T{sub 4}-glucuronidation activity, but significant increases in hepatic Ugt1a1, Ugt1a7, and Ugt2b5 mRNA expression accompany significant decreases in T{sub 4} concentrations at 100 mg/kg/day of BDE 47. Induction of PROD activity occurred at the lowest dose (3 mg/kg/day). Cyp2b10 mRNA expression also increased significantly at 10 and 100 mg/kg/day. These key findings show that BDE activates the nuclear receptor, CAR. Decreases in Mdr1a mRNA expression also occurred at the lowest dose administered (3 mg/kg/day BDE 47). BDE 47 exposure also decreased hepatic transthyretin and monocarboxylate transporter 8 (Mct8) mRNA expression, suggesting that while induction of UGTs may be partly responsible for T{sub 4} decreases, other mechanisms are also involved. No effects were seen in the kidney. We conclude that changes in hepatic UGTs and transporters may be involved in decreases in circulating T{sub 4} following BDE 47 exposure.

  7. Possible mechanisms of thyroid hormone disruption in mice by BDE 47, a major polybrominated diphenyl ether congener.

    PubMed

    Richardson, Vicki M; Staskal, Daniele F; Ross, David G; Diliberto, Janet J; DeVito, Michael J; Birnbaum, Linda S

    2008-02-01

    Polybrominated diphenyl ethers (PBDEs) are a class of polyhalogenated aromatic compounds commercially used as fire retardants in consumer products. These compounds have been shown to decrease thyroid hormone concentrations in rodents after acute exposures. This study examines the ability of 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) to decrease circulating thyroid hormone concentrations and pairs this with BDE 47-induced effects on genes involved in thyroid hormone homeostasis. Female C57BL/6 mice (9 weeks old) were orally administered 3, 10, or 100 mg/kg/day of BDE 47 for 4 days. Animals were euthanized 24 h after the final dose (day 5) and liver, kidney, and serum were collected for analysis. BDE 47 caused a significant 43% decrease at 100 mg/kg/day in serum total thyroxine (T(4)) concentrations. There was no increase in hepatic T(4)-glucuronidation activity, but significant increases in hepatic Ugt1a1, Ugt1a7, and Ugt2b5 mRNA expression accompany significant decreases in T(4) concentrations at 100 mg/kg/day of BDE 47. Induction of PROD activity occurred at the lowest dose (3 mg/kg/day). Cyp2b10 mRNA expression also increased significantly at 10 and 100 mg/kg/day. These key findings show that BDE activates the nuclear receptor, CAR. Decreases in Mdr1a mRNA expression also occurred at the lowest dose administered (3 mg/kg/day BDE 47). BDE 47 exposure also decreased hepatic transthyretin and monocarboxylate transporter 8 (Mct8) mRNA expression, suggesting that while induction of UGTs may be partly responsible for T(4) decreases, other mechanisms are also involved. No effects were seen in the kidney. We conclude that changes in hepatic UGTs and transporters may be involved in decreases in circulating T(4) following BDE 47 exposure.

  8. Novel PKCη Is Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

    PubMed Central

    Lachmann, Sylvie; Rommeleare, Jean; Nüesch, Jürg P. F.

    2003-01-01

    The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCη in the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle. PMID:12829844

  9. Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

    PubMed

    Hajjar, Adeline M; Ernst, Robert K; Fortuno, Edgardo S; Brasfield, Alicia S; Yam, Cathy S; Newlon, Lindsay A; Kollmann, Tobias R; Miller, Samuel I; Wilson, Christopher B

    2012-01-01

    Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

  10. Electroretinography of wild-type and Cry mutant mice reveals circadian tuning of photopic and mesopic retinal responses.

    PubMed

    Cameron, Morven A; Barnard, Alun R; Hut, Roelof A; Bonnefont, Xavier; van der Horst, Gijsbertus T J; Hankins, Mark W; Lucas, Robert J

    2008-12-01

    Attempts to understand circadian organization in the mammalian retina have concentrated increasingly on the mouse. However, rather little is known regarding circadian control of retinal light responses in this species. Here, the authors address this deficit using electroretinogram (ERG) recordings in C57BL/6 mice to evaluate rhythmicity in the wild-type retina and to identify the consequences of circadian clock loss in Cry1(- /-)Cry2(-/-) mice. They observe a circadian rhythm in the ERG waveform under light-adapted, cone-isolating conditions in wild-type mice, with b-wave speed and amplitude and the total power of oscillatory potentials all enhanced during the day. Wild types also exhibited a circadian dependence to ERG amplitude under dark-adapted conditions, but only when the flash stimulus was sufficiently bright to lie within the response range of cones. Cry1(-/ -)Cry2(-/-) mice lacked rhythmicity but retained superficially normal ERGs under all conditions suggesting that circadian clocks are dispensable for general retinal function. However, clock loss was associated with subtle abnormalities in retinal responses, with the amplitude of cone and mixed rod + cone ERGs constitutively enhanced. These data suggest that circadian clocks drive a fundamental fine-tuning of retinal pathways that is particularly apparent under conditions in which vision relies upon either cones alone or mixed rod + cone photoreception.

  11. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    SciTech Connect

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-05-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group).

  12. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice.

    PubMed

    Bennewitz, Margaret F; Watkins, Simon C; Sundd, Prithu

    2014-07-07

    Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus.

  13. Interrelationship between lymphocytes and leptin in fat depots of obese mice revealed by changes in nutritional status.

    PubMed

    Lolmède, Karine; Zakaroff-Girard, Alexia; Dray, Cedric; Renoud, Marie-Laure; Daviaud, Danièle; Burcelin, Rémy; Lafontan, Max; Galitzky, Jean; Bouloumié, Anne

    2015-09-01

    The mechanisms underlying the relationships between nutritional status and immunity remain to be fully characterized. The present study was undertaken to analyze by flow cytometry, in the context of diet-induced obesity, the status of immune cells in subcutaneous, and epididymal fat depots in wild-type and immunodeficient Rag2-/- mice submitted to nutritional challenge, i.e., 48-h fasting and 1-week refeeding. In parallel, the responsiveness of mature adipocytes and immune cells in bone marrow, lymph node, and liver were also analyzed. The results show that fasting in obese wild-type mice induces a prominent lipolysis in epididymal AT and immunosuppression restricted to both subcutaneous and epididymal AT, characterized by reduced number of CD4+ T and B lymphocytes and M1/M2 macrophages associated with reduced leptin and increased FGF21 expression in mature adipocytes. One-week refeeding was sufficient to reverse the fasting-induced effects. Obese immunodeficient mice under nutritional challenge exhibited no changes in adipocyte leptin expression and no marked trafficking of AT macrophages or NK cells, while the fasted-induced upregulation of FGF21 expression was maintained as well as the lipolytic responses. The present results demonstrate that, in a context of diet-induced obesity, fasting-induced immunosuppression is restricted to fat depots in immunocompetent mice. Lack of adipocyte leptin regulation and fasting-induced immunosuppression in obese immunodeficient mice strongly suggests that lymphocytes are involved in the modulation of adipocyte leptin expression on one hand and on the other that leptin is involved in the immune changes in AT according to nutritional status.

  14. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice

    PubMed Central

    Bennewitz, Margaret F; Watkins, Simon C; Sundd, Prithu

    2014-01-01

    Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus. PMID:25995970

  15. Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold

    PubMed Central

    Drennan, Catherine L.; Nonato, M. Cristina

    2016-01-01

    Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases. PMID:27528683

  16. Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

    PubMed

    Feliciano, Patricia R; Drennan, Catherine L; Nonato, M Cristina

    2016-08-30

    Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases.

  17. Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

    PubMed Central

    Sawitzky, Mandy; Zeissler, Anja; Langhammer, Martina; Bielohuby, Maximilian; Stock, Peggy; Hammon, Harald M.; Görs, Solvig; Metges, Cornelia C.; Stoehr, Barbara J. M.; Bidlingmaier, Martin; Fromm-Dornieden, Carolin; Baumgartner, Bernhard G.; Christ, Bruno; Brenig, Bertram; Binder, Gerhard; Metzger, Friedrich; Renne, Ulla; Hoeflich, Andreas

    2012-01-01

    We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice. PMID:22768110

  18. Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice

    PubMed Central

    Nishimune, Hiroshi; Badawi, Yomna; Mori, Shuuichi; Shigemoto, Kazuhiro

    2016-01-01

    Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography. PMID:27321892

  19. Bone marrow transplantation across major histocompatibility barriers in mice. II. T cell requirement for engraftment in total lymphoid irradiation-conditioned recipients

    SciTech Connect

    Vallera, D.A.; Soderling, C.C.; Carlson, G.J.; Kersey, J.H.

    1982-03-01

    Studies were undertaken to examine the role of T lymphocytes in engraftment of bone marrow (BM) in animals conditioned with total lymphoid irradiation (TLI) prior to transplantation across major histocompatibility barriers. Donor BM (added as a source of lymphohematopoietic stem cells) and spleen cells (added as a source of graft-versus-host disease (GVHD)-causing cells) were pretreated in vitro with monoclonal anti-Thy-1.2 plus complement (C). T cell-depleted grafts were then give to allogeneic mice conditioned with 900 rad of single dose TLI plus cyclophosphamide (CY). These mice did not engraft. Even in the absence of added spleen cells, elimination of the small T cell population from donor BM grafts prevented engraftment compared with animals that received the same conditioning regimen and untreated donor cells. These control animals demonstrated uniform evidence of engraftment about 1 month after transplantation. Similar findings were reported when recipients were conditioned with fractionated 17 x 200-rad TLI. In TLI plus CY-conditional recipients, we have also observed that increasing the donation of treated bone marrow cells still did not result in significant engraftment. Furthermore, graft failure in mice receiving normal dosages of anti-Thy-1.2 plus C-treated donor cells was not a strain-restricted phenomenon. Moreover, removal of bone marrow T cells with monoclonal anti-Lyt-1 plus complement also resulted in graft failure in TLI-conditioned recipients. In contrast to TLI conditioning, when Thy-1.2 plus C-treated donor cells were given to recipients conditioned with total body irradiation (TBI), a high percentage of engraftment was demonstrated by an H-2 microcytotoxicity assay. Plausible mechanisms for there findings are discussed.

  20. Leoligin, the major lignan from Edelweiss, inhibits 3-hydroxy-3-methyl-glutaryl-CoA reductase and reduces cholesterol levels in ApoE-/- mice.

    PubMed

    Scharinger, Bernhard; Messner, Barbara; Türkcan, Adrian; Schuster, Daniela; Vuorinen, Anna; Pitterl, Florian; Heinz, Katharina; Arnhard, Kathrin; Laufer, Günther; Grimm, Michael; Stuppner, Hermann; Oberacher, Herbert; Eller, Philipp; Ritsch, Andreas; Bernhard, David

    2016-10-01

    The health benefit through the control of lipid levels in hyperlipidaemic individuals is evident from a large number of studies. The pharmacological options to achieve this goal shall be as specific and personalized as the reasons for and co-factors of hyperlipidaemia. It was the goal of this study to reveal the impact of leoligin on cholesterol levels and to define its mechanism of action. Oral application of leoligin in ApoE-/- mice led to significantly reduced total serum cholesterol levels and a reduction in postprandial blood glucose peak levels. In the absence of biochemical signs of toxicity, leoligin treatment resulted in reduced weight gain in mice. The effects of leoligin on serum cholesterol levels may be due to a direct inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) by a unique, non-statin-like binding mode. Postprandial serum glucose peaks may be reduced by a mild peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonistic activity of leoligin. No effect on atherosclerotic plaque size was observed. As a non-toxic, cholesterol-, peak glucose-, and weight gain-lowering compound, leoligin continues to fulfil characteristics of a potential agent for the treatment of cardiovascular disease (CVD). The counterregulatory overexpression of hepatic HMGCR in leoligin treated animals possibly explains the missing permanent anti-atherosclerotic effect.

  1. Metabolomics reveals significant impairments in the immune system of the APP/PS1 transgenic mice of Alzheimer's disease.

    PubMed

    González-Domínguez, Raúl; García-Barrera, Tamara; Vitorica, Javier; Gómez-Ariza, José Luis

    2015-02-01

    Inflammatory processes and other failures related to the immune system are common features associated with Alzheimer's disease (AD), in both brain and the peripheral system. Thus, the study of the main organs of the immune system may have a great potential for the elucidation of pathological mechanisms underlying these abnormalities. This is the first metabolomic investigation performed in spleen and thymus from transgenic mice of AD. Tissues were fingerprinted using a metabolomic platform comprising GC-MS and ultra-HPLC-MS. Multivariate statistics demonstrated significant differences in numerous metabolites between the APP/PS1 mice and wild-type controls, and it was proven that multiple biochemical pathways are disturbed in these organs including abnormal metabolism of phospholipids, energy deficiencies, altered homeostasis of amino acids, oxidative stress, and others. Therefore, these findings highlight the importance of the proper metabolic functioning of peripheral immune system in the development of neurodegenerative disorders such as AD.

  2. Topological analyses in APP/PS1 mice reveal that astrocytes do not migrate to amyloid-β plaques

    PubMed Central

    Galea, Elena; Morrison, Will; Hudry, Eloise; Arbel-Ornath, Michal; Bacskai, Brian J.; Gómez-Isla, Teresa; Stanley, H. Eugene; Hyman, Bradley T.

    2015-01-01

    Although the clustering of GFAP immunopositive astrocytes around amyloid-β plaques in Alzheimer’s disease has led to the widespread assumption that plaques attract astrocytes, recent studies suggest that astrocytes stay put in injury. Here we reexamine astrocyte migration to plaques, using quantitative spatial analysis and computer modeling to investigate the topology of astrocytes in 3D images obtained by two-photon microscopy of living APP/PS1 mice and WT littermates. In WT mice, cortical astrocyte topology fits a model in which a liquid of hard spheres exclude each other in a confined space. Plaques do not disturb this arrangement except at very large plaque loads, but, locally, cause subtle outward shifts of the astrocytes located in three tiers around plaques. These data suggest that astrocytes respond to plaque-induced neuropil injury primarily by changing phenotype, and hence function, rather than location. PMID:26644572

  3. Bone marrow transplantation across major histocompatibility barriers in mice: II. T cell requirement for engraftment in total lymphoid irradiation-conditioned recipients

    SciTech Connect

    Vallera, D.A.; Soderling, C.C.B.; Carlson, G.J.; Kersey, J.H.

    1982-03-01

    Studies were undertaken to examine the role of T lymphocytes in engraftment of bone marrow (BM) in animals conditioned with total lymphoid irradiation (TLI) prior to transplantation across major histocompatability barriers.Donor BM (added as a source of lymphohematopoietic stem cells) and spleen cells (added as a source of graft-versus-host disease (GVHD)-causing cells) were pretreated in vitro with monoclonal anti-Thy-1.2 plus complement (C). T cell-depleted grafts were then given to allogeneic mice conditioned with 900 rad of single dose TLI plus cyclophosphamide (CY). These mice did not engraft. Even in the absence of added spleen cells, elimination of the small T cell population from donor BM grafts prevented engraftment compared with animals that received the same conditioning regimen and untreated donor cells. These control animals demonstrated uniform evidence of engraftment about 1 month after transplantation. Similar findings were reported when recipients were conditioned with fractionated 17 x 100-rad TLI. In TLI plus CY-conditioned recipients, it was also observed that increasing the donation of treated bone marrow cells still did not result in significant engraftment. In contrast to TLI conditioning, when Thy-1.2 plus C-treated donor cells were given to recipients conditioned with total body irradiation (TBI), a high percentage of engraftment was demonstrated by an H-2 microcytotoxicity assay. Plausible mechanisms for these findings are discussed. (JMT)

  4. ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation

    PubMed Central

    Romano, Rose-Anne; Smalley, Kirsten; Magraw, Caitlin; Serna, Vanida Ann; Kurita, Takeshi; Raghavan, Srikala; Sinha, Satrajit

    2012-01-01

    The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ΔNp63. To investigate the role of ΔNp63 in epidermal morphogenesis we generated ΔNp63 knock-in mice in which the ΔNp63-specific exon is replaced by GFP. Homozygous ΔNp63gfp/gfp animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. ΔNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of ΔNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to ΔNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of ΔNp63 as demonstrated by ChIP experiments. The analysis of ΔNp63gfp/gfp knockout mice reaffirms the indispensable role of the ΔN isoform of p63 in epithelial biology and confirms that ΔNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate. PMID:22274697

  5. Stimulation of cardiomyocyte regeneration in neonatal mice and in human myocardium with neuregulin reveals a therapeutic window

    PubMed Central

    Polizzotti, Brian D.; Ganapathy, Balakrishnan; Walsh, Stuart; Choudhury, Sangita; Ammanamanchi, Niyatie; Bennett, David G.; dos Remedios, Cristobal G.; Haubner, Bernhard J.; Penninger, Josef M.; Kuhn, Bernhard

    2015-01-01

    Background Pediatric patients with heart failure are treated with medical therapies that were developed for adult patients. These therapies have been shown to be ineffective in pediatric trials, leading to the recognition that new pediatric-specific therapies must be developed. We have previously shown that administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates heart muscle cell (cardiomyocyte) regeneration in adult mice. We hypothesized that rNRG1 administration may be more effective in the neonatal period, which could provide a new therapeutic paradigm for treating heart failure in pediatric patients. Methods We used a cryoinjury model to induce myocardial dysfunction and scar formation for evaluating the effectiveness of rNRG1-administration in neonatal mice. We evaluated the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. Results After cryoinjury in neonatal mice, early administration of rNRG1 from birth for 34 days improved myocardial function and reduced the prevalence of transmural scars. In contrast, late administration of rNRG1 from 4 to 34 days after cryoinjury transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease less than 6 months of age. Conclusion Our results identify a more effective time period within which to execute future clinical trials of rNRG1 for stimulating cardiomyocyte regeneration. PMID:25834111

  6. Molecular regulation of sexual preference revealed by genetic studies of 5-HT in the brains of male mice.

    PubMed

    Liu, Yan; Jiang, Yun'ai; Si, Yunxia; Kim, Ji-Young; Chen, Zhou-Feng; Rao, Yi

    2011-04-07

    Although the question of to whom a male directs his mating attempts is a critical one in social interactions, little is known about the molecular and cellular mechanisms controlling mammalian sexual preference. Here we report that the neurotransmitter 5-hydroxytryptamine (5-HT) is required for male sexual preference. Wild-type male mice preferred females over males, but males lacking central serotonergic neurons lost sexual preference although they were not generally defective in olfaction or in pheromone sensing. A role for 5-HT was demonstrated by the phenotype of mice lacking tryptophan hydroxylase 2 (Tph2), which is required for the first step of 5-HT synthesis in the brain. Thirty-five minutes after the injection of the intermediate 5-hydroxytryptophan (5-HTP), which circumvented Tph2 to restore 5-HT to the wild-type level, adult Tph2 knockout mice also preferred females over males. These results indicate that 5-HT and serotonergic neurons in the adult brain regulate mammalian sexual preference.

  7. CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes

    PubMed Central

    Zhong, Hua; Chen, Yiyun; Li, Yumei; Chen, Rui; Mardon, Graeme

    2015-01-01

    The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Here, we utilized the CRISPR-Cas9 system to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, mutations of which are thought to cause Leber congenital amaurosis (LCA), an early-onset form of blindness. We found that complete loss of Kcnj13 is likely postnatal lethal. Among surviving F0-generation mice examined, 80% show mosaic KCNJ13 expression in the retinal pigment epithelium (RPE). Mosaic expression correlates with decreased response to light and photoreceptor degeneration, indicating that Kcnj13 mutant mice mimic human KCNJ13-related LCA disease. Importantly, mosaic animals enable us to directly compare Kcnj13 mutant and wild-type RPE cells in the same eye. We found that RPE cells lacking KCNJ13 protein still survive but overlying photoreceptors exhibit cell degeneration. At the same time, wild-type RPE cells can rescue neighboring photoreceptor cells that overlie mutant RPE cells. These results suggest that KCNJ13 expression is required for RPE cells to maintain photoreceptor survival. Moreover, we show that CRISPR-Cas9 engineered mosaicism can be used to rapidly test candidate gene function in vivo. PMID:25666713

  8. Natural History of Meningioma Development in Mice Reveals: A Synergy of Nf2 and p16Ink4a Mutations

    PubMed Central

    Kalamarides, Michel; Stemmer-Rachamimov, Anat O; Takahashi, Masaya; Han, Zhi-Yan; Chareyre, Fabrice; Niwa-Kawakita, Michiko; Black, Peter M; Carroll, Rona S; Giovannini, Marco

    2008-01-01

    Meningiomas account for approximately 30% of all primary central nervous system tumors and are found in half of neurofibromatosis type 2 patients often causing significant morbidity. Although most meningiomas are benign, 10% are classified as atypical or anaplastic, displaying aggressive clinical behavior. Biallelic inactivation of the neurofibromatosis 2 (NF2) tumor suppressor is associated with meningioma formation in all NF2 patients and 60% of sporadic meningiomas. Deletion of the p16INK4a/p14ARF locus is found in both benign and malignant meningiomas, while mutation of the p53 tumor suppressor gene is uncommon. Previously, we inactivated Nf2 in homozygous conditional knockout mice by adenoviral Cre delivery and showed that Nf2 loss in arachnoid cells is rate-limiting for meningioma formation. Here, we report that additional nullizygosity for p16Ink4a increases the frequency of meningioma and meningothelial proliferation in these mice without modifying the tumor grade. In addition, by using magnetic resonance imaging (MRI) to screen a large cohort of mutant mice, we were able to detect meningothelial proliferation and meningioma development opening the way to future studies in which therapeutic interventions can be tested as preclinical assessment of their potential clinical application. PMID:17924978

  9. Intranasal siRNA administration reveals IGF2 deficiency contributes to impaired cognition in Fragile X syndrome mice

    PubMed Central

    Pardo, Marta; Cheng, Yuyan; Velmeshev, Dmitry; Magistri, Marco; Martinez, Ana; Faghihi, Mohammad A.; Jope, Richard S.; Beurel, Eleonore

    2017-01-01

    Molecular mechanisms underlying learning and memory remain imprecisely understood, and restorative interventions are lacking. We report that intranasal administration of siRNAs can be used to identify targets important in cognitive processes and to improve genetically impaired learning and memory. In mice modeling the intellectual deficiency of Fragile X syndrome, intranasally administered siRNA targeting glycogen synthase kinase-3β (GSK3β), histone deacetylase-1 (HDAC1), HDAC2, or HDAC3 diminished cognitive impairments. In WT mice, intranasally administered brain-derived neurotrophic factor (BDNF) siRNA or HDAC4 siRNA impaired learning and memory, which was partially due to reduced insulin-like growth factor-2 (IGF2) levels because the BDNF siRNA– or HDAC4 siRNA–induced cognitive impairments were ameliorated by intranasal IGF2 administration. In Fmr1–/– mice, hippocampal IGF2 was deficient, and learning and memory impairments were ameliorated by IGF2 intranasal administration. Therefore intranasal siRNA administration is an effective means to identify mechanisms regulating cognition and to modulate therapeutic targets. PMID:28352664

  10. Functional interplay between cylindromatosis and histone deacetylase 6 in ciliary homeostasis revealed by phenotypic analysis of double knockout mice

    PubMed Central

    Ran, Jie; Yu, Fan; Qin, Juan; Zhang, Yijun; Yang, Yunfan; Li, Dengwen; Zhou, Jun; Liu, Min

    2016-01-01

    Cilia are present in most vertebrate tissues with a wide variety o