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Sample records for mice reveals major

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  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. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  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. 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

  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. 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.

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

    DTIC Science & Technology

    2012-06-20

    Mustafa Köksal, Jesusa Josue, Kunio Yamazaki, Gary K. Beauchamp Monell Chemical Senses Center 3500 Market Street Philadelphia, PA 19104 -3360 REPORT...Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA b Human Signatures Branch, Forecasting Division, Human Effectiveness...www.informatics.jax.org). Individual urine-based olfactory signatures are not an exception. Mice can discriminate the urinary odorants of other mice with different

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  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. 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

  15. 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.

  16. 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.

  17. 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

    2017-02-22

    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.

  18. Major urinary protein 5, a scent communication protein, is regulated by dietary restriction and subsequent re-feeding in mice

    PubMed Central

    Giller, K.; Huebbe, P.; Doering, F.; Pallauf, K.; Rimbach, G.

    2013-01-01

    Major urinary proteins (Mups) are important for rodent scent communication and sexual behaviour. Recent evidence suggests that Mup1 may be regulated by fasting and re-feeding (RF). However, other Mup isoforms are poorly investigated, and data on the impact of long-term dietary restriction (DR) and ad libitum RF on Mup expression are missing. We investigated the effects of long-term 25 per cent DR and subsequent RF on Mup expression in male C57BL6 mice. DR significantly decreased Mup gene expression, hepatic and urinary protein levels compared with ad libitum (AL) fed control mice, with the greatest downregulation found for Mup5 expression. The decline in Mup expression was inverted by six months of RF. Because of inhibitory glucocorticoid response elements in the genomic sequence of the Mup5 gene, the observed inverse correlation of nuclear glucocorticoid receptor levels with Mup expression in response to DR and subsequent RF is a possible regulatory mechanism. Additionally, gene-expression-inhibiting histone deacetylation (H3K9) occurred in the region of the Mup5 gene in response to DR. We assume that Mup may act as a molecular switch linking nutritional status to sexual behaviour of mice, and thereby regulating male fertility and reproduction in response to food supply. PMID:23446533

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  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. 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

  6. Leishmania major lacking arginase are auxotrophic for polyamines but retain infectivity to susceptible BALB/c mice

    PubMed Central

    Reguera, Rosa M.; Balaña-Fouce, Rafael; Showalter, Melissa; Hickerson, Suzanne; Beverley, Stephen M.

    2009-01-01

    Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from L-ornithine has been well studied in protozoa, the metabolic origin(s) of L-ornithine have received less attention. Arginase (EC 3.5.3.1) catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg− knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or L-ornithine for growth. A complemented line (arg−/+ARG) expressing arginase from a multicopy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors α-difluoromethylornithine (DFMO) and Nω-hydroxy-L-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg− parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of L-arginine metabolism in both pro- and anti-parasitic responses involving host nitric

  7. 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.

  8. 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

  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. 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.

  11. 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

  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. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  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. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Infection with arginase deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis1

    PubMed Central

    Muleme, Helen M; Reguera, Rosa M; Berard, Alicia; Azinwi, Richard; Jia, Ping; Okwor, Ifeoma B; Beverley, Stephen; Uzonna, Jude E

    2009-01-01

    The balance between the products of L-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. L-arginine can be oxidized by host inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), which contributes to parasite killing. In contrast, L-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considereable potential to modulate infectivity and disease pathogenesis. Here, we compare the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg-). We found that arg- L. major are impaired in their macrophage infectivity in vitro independent of host iNOS activities. As with in vitro results, the proliferation of arg- L. major in animal infections was also significantly impaired in vivo resulting in delayed onset of lesion development, attenuated pathology and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg- L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg- than in WT infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities leading and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity. PMID:19923451

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  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. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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).

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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)

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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 of functions, and abnormalities of cilia are linked to numerous human disorders. However, the molecular events underlying ciliary homeostasis are poorly understood. In this study, we generated double knockout (DKO) mice for the deubiquitinase cylindromatosis (CYLD) and histone deacetylase 6 (HDAC6), two critical ciliary regulators. The Cyld/Hdac6 DKO mice were phenotypically normal and showed no obvious variances in weight or behavior compared with their wild-type littermates. Strikingly, Cyld loss-induced ciliary defects in the testis, trachea, and kidney were abrogated in the Cyld/Hdac6 DKO mice. In addition, the diminished α-tubulin acetylation and impaired sonic hedgehog signaling caused by loss of Cyld were largely restored by simultaneous deletion of Hdac6. We further found by immunofluorescence microscopy a colocalization of CYLD and HDAC6 at the centrosome/basal body and, interestingly, loss of Cyld promoted the localization of HDAC6 at the centrosome/basal body. These findings provide physiological insight into the ciliary role of the CYLD/HDAC6 axis and suggest a functional interplay between these two proteins in ciliary homeostasis. PMID:27028867

  17. The major acute-phase protein, serum amyloid P component, in mice is not involved in endogenous resistance against tumor necrosis factor alpha-induced lethal hepatitis, shock, and skin necrosis.

    PubMed

    Van Molle, W; Hochepied, T; Brouckaert, P; Libert, C

    2000-09-01

    The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) induces lethal hepatitis when injected into D-(+)-galactosamine-sensitized mice on the one hand or systemic inflammatory response syndrome (SIRS) in normal mice on the other hand. We studied whether serum amyloid P component (SAP), the major acute-phase protein in mice, plays a protective role in both lethal models. For this purpose, we used SAP(0/0) mice generated by gene targeting. We studied the lethal response of SAP(0/0) or SAP(+/+) mice to both lethal triggers but found no differences in the sensitivity of both types of mice. We also investigated whether SAP is involved in establishing two types of endogenous protection: one using a single injection of interleukin-1beta (IL-1beta) for desensitization and clearly involving a liver protein, the other by tolerizing mice for 5 days using small doses of human TNF-alpha. Although after IL-1beta or after tolerization the SAP levels in the serum had risen fourfold in the control mice and not in the SAP(0/0) mice, the same extents of desensitization and tolerization were achieved. Finally, we observed that the induction of hemorrhagic necrosis in the skin of mice by two consecutive local injections with TNF-alpha was not altered in SAP(0/0) mice. We conclude that the presence or absence of SAP has no influence on the sensitivity of mice to TNF-alpha-induced hepatitis, SIRS, and hemorrhagic necrosis or on the endogenous protective mechanisms of desensitization or tolerization.

  18. Differential potassium channel gene regulation in BXD mice reveals novel targets for pharmacogenetic therapies to reduce heavy alcohol drinking.

    PubMed

    Rinker, Jennifer A; Fulmer, Diana B; Trantham-Davidson, Heather; Smith, Maren L; Williams, Robert W; Lopez, Marcelo F; Randall, Patrick K; Chandler, L Judson; Miles, Michael F; Becker, Howard C; Mulholland, Patrick J

    2017-02-01

    Alcohol (ethanol) dependence is a chronic relapsing brain disorder partially influenced by genetics and characterized by an inability to regulate harmful levels of drinking. Emerging evidence has linked genes that encode KV7, KIR, and KCa2 K(+) channels with variation in alcohol-related behaviors in rodents and humans. This led us to experimentally test relations between K(+) channel genes and escalation of drinking in a chronic-intermittent ethanol (CIE) exposure model of dependence in BXD recombinant inbred strains of mice. Transcript levels for K(+) channel genes in the prefrontal cortex (PFC) and nucleus accumbens (NAc) covary with voluntary ethanol drinking in a non-dependent cohort. Transcripts that encode KV7 channels covary negatively with drinking in non-dependent BXD strains. Using a pharmacological approach to validate the genetic findings, C57BL/6J mice were allowed intermittent access to ethanol to establish baseline consumption before they were treated with retigabine, an FDA-approved KV7 channel positive modulator. Systemic administration significantly reduced drinking, and consistent with previous evidence, retigabine was more effective at reducing voluntary consumption in high-drinking than low-drinking subjects. We evaluated the specific K(+) channel genes that were most sensitive to CIE exposure and identified a gene subset in the NAc and PFC that were dysregulated in the alcohol-dependent BXD cohort. CIE-induced modulation of nine genes in the NAc and six genes in the PFC covaried well with the changes in drinking induced by ethanol dependence. Here we identified novel candidate genes in the NAc and PFC that are regulated by ethanol dependence and correlate with voluntary drinking in non-dependent and dependent BXD mice. The findings that Kcnq expression correlates with drinking and that retigabine reduces consumption suggest that KV7 channels could be pharmacogenetic targets to treat individuals with alcohol addiction.

  19. Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

    PubMed Central

    Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

    2016-01-01

    ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA

  20. Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or beta-adrenergic receptors triggers cellular response in mice.

    PubMed Central

    Solano, A R; Cremaschi, G; Sánchez, M L; Borda, E; Sterin-Borda, L; Podestá, E J

    1988-01-01

    Purified IgG from BALB/c mouse anti-C3H serum exerts positive inotropic and chronotropic effects in C3H mouse atria and induces testosterone synthesis in C3H mouse Leydig cells. The effect depends on IgG concentration and can be abolished by beta-adrenergic-receptor and luteinizing hormone-receptor antagonists. IgG interferes with the binding of dihydroalprenolol and luteinizing hormone. Monoclonal antibodies against major histocompatibility complex class I antigens were active on the Leydig cells of C3H and BALB/c mice. There was a parallelism between the effect of each individual monoclonal antibody with specificity for a particular haplotype and the response of the target cell from the strains carrying such haplotypes. These antibodies could precipitate the soluble luteinizing hormone-receptor complex. The results suggested that bound hormone triggers the association of major histocompatibility class I antigen with the receptor, thereby activating the respective target cells. PMID:2839829

  1. Green tea polyphenol (−)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: Responses of major antioxidant enzymes and the Nrf2 rescue pathway

    SciTech Connect

    Wang, Dongxu; Wang, Yijun; Wan, Xiaochun; Yang, Chung S.; Zhang, Jinsong

    2015-02-15

    (−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been suggested to have numerous health-promoting effects. On the other hand, high-dose EGCG is able to evoke hepatotoxicity. In the present study, we elucidated the responses of hepatic major antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2) rescue pathway to high-dose levels of EGCG in Kunming mice. At a non-lethal toxic dose (75 mg/kg, i.p.), repeated EGCG treatments markedly decreased the levels of superoxide dismutase, catalase, and glutathione peroxidase. As a rescue response, the nuclear distribution of Nrf2 was significantly increased; a battery of Nrf2-target genes, including heme oxygenase 1 (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and those involved in glutathione and thioredoxin systems, were all up-regulated. At the maximum tolerated dose (45 mg/kg, i.p.), repeated EGCG treatments did not disturb the major antioxidant defense. Among the above-mentioned genes, only HO1, NQO1, and GST genes were significantly but modestly up-regulated, suggesting a comprehensive and extensive activation of Nrf2-target genes principally occurs at toxic levels of EGCG. At a lethal dose (200 mg/kg, i.p.), a single EGCG treatment dramatically decreased not only the major antioxidant defense but also the Nrf2-target genes, demonstrating that toxic levels of EGCG are able to cause a biphasic response of Nrf2. Overall, the mechanism of EGCG-triggered hepatotoxicity involves suppression of major antioxidant enzymes, and the Nrf2 rescue pathway plays a vital role for counteracting EGCG toxicity. - Highlights: • EGCG at maximum tolerated dose does not disturb hepatic major antioxidant defense. • EGCG at maximum tolerated dose modestly upregulates hepatic Nrf2 target genes. • EGCG at toxic dose suppresses hepatic major antioxidant enzymes. • EGCG at non-lethal toxic dose pronouncedly activates hepatic Nrf2 rescue response. • EGCG at

  2. Transcriptional analysis reveals a high impact of conjugated linoleic acid on stearoyl-Coenzyme A desaturase 1 mRNA expression in mice gastrocnemius muscle.

    PubMed

    Parra, Pilar; Serra, Francisca; Palou, Andreu

    2012-10-01

    We examined the potential implication of skeletal muscle in the fat-lowering effect observed in mice treated with moderate doses of CLA. In experiment 1, mice fed with a standard-fat diet were orally treated with sunflower oil (control) and 3 or 10 mg CLA mixture/day for 37 days. In experiment 2, mice were fed with a high-fat diet for 65 days. For the first 30 days, they received the same doses as in experiment 1 and, from that time onwards, animals received double doses. Gene expression of key proteins involved in fatty acid transport, oxidation, regulation of lipid and carbohydrate utilization, composition of muscle fiber, and thermogenesis were determined and, in most of them, no major impact of CLA was seen. Therefore, enhancement of fatty acid oxidation in muscle did not seem to contribute to the antiobesity effect of CLA as seen in other studies with higher CLA doses. However, a strong induction of classically associated lipogenic genes such as Fasn (up to twofold) and, particularly, Scd1 (up to ninefold) was found. This activation could contribute to a protective role in muscle cells, since expression of ER stress markers was decreased and inversely correlated with the induction of Scd1.

  3. Genomic Profiling Reveals Unique Molecular Alterations in Hepatoblastomas and Adjacent Hepatocellular Carcinomas in B6C3F1 Mice.

    PubMed

    Bhusari, Sachin; Pandiri, Arun R; Nagai, Hiroaki; Wang, Yu; Foley, Julie; Hong, Hue-Hua L; Ton, Thai-Vu; DeVito, Michael; Shockley, Keith R; Peddada, Shyamal D; Gerrish, Kevin E; Malarkey, David E; Hooth, Michelle J; Sills, Robert C; Hoenerhoff, Mark J

    2015-12-01

    The cell of origin of hepatoblastoma (HB) in humans and mice is unknown; it is hypothesized to be a transformed hepatocyte, oval cell, or hepatic progenitor cell. In mice, current dogma is that HBs arise from preexisting hepatocellular neoplasms as a result of further neoplastic transformation. However, there is little evidence supporting this direct relationship. To better understand the relationship between hepatocellular carcinoma (HCC) and HB and determine molecular similarities between mouse and human HB, global gene expression analysis and targeted mutation analysis were performed using HB, HCC, and adjacent liver from the same animals in a recent National Toxicology Program bioassay. There were significant differences in Hras and Ctnnb1 mutation spectra, and by microarray, HBs showed dysregulation of embryonic development, stem cell pluripotency, and genomic imprinting compared to HCC. Meta-analysis showed similarities between HB, early mouse embryonic liver, and hepatocyte-derived stem/progenitor cells compared to HCC. Our data show that there are striking differences between HB and HCC and suggest that HB is a significantly different entity that may arise from a hepatic precursor cell. Furthermore, mouse HB is similar to the human disease at the pathway level and therefore is likely a relevant model for evaluating human cancer hazard.

  4. RNA-sequencing analysis reveals the hepatotoxic mechanism of perfluoroalkyl alternatives, HFPO2 and HFPO4, following exposure in mice.

    PubMed

    Wang, Jianshe; Wang, Xiaoyang; Sheng, Nan; Zhou, Xiujuan; Cui, Ruina; Zhang, Hongxia; Dai, Jiayin

    2017-04-01

    The toxicological impact of traditional perfluoroalkyl chemicals has led to the elimination and restriction of these substances. However, many novel perfluoroalkyl alternatives remain unregulated and little is known about their potential effects on environmental and human health. Daily administration of two alternative perfluoroalkyl substances, HFPO2 and HFPO4 (1 mg kg(-1) body weight), for 28 days resulted in hepatomegaly and hepatic histopathological injury in mice, particularly in the HFPO4 group. We generated and compared high-throughput RNA-sequencing data from hepatic tissues in control and treatment group mice to clarify the mechanism of HFPO2 and HFPO4 hepatotoxicity. We identified 146 (101 upregulated, 45 downregulated) and 1295 (716 upregulated, 579 downregulated) hepatic transcripts that exhibited statistically significant changes (fold change ≥2 or ≤0.5, false discovery rate < 0.05) after HFPO2 and HFPO4 treatment, respectively. Among them, 111 (82 upregulated, 29 downregulated) transcripts were changed in both groups, and lipid metabolism associated genes were dominant. Thus, similar to their popular predecessors, HFPO2 and HFPO4 exposure exerted hepatic effects, including hepatomegaly and injury, and altered lipid metabolism gene levels in the liver, though HFPO4 exerted greater hepatotoxicity than HFPO2. The unregulated use of these emerging perfluoroalkyl alternatives may affect environmental and human health, and their biological effects need further exploration. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice.

    PubMed

    Chatel, Jean-Marc; Nouaille, Sebastien; Adel-Patient, Karine; Le Loir, Yves; Boe, Herman; Gruss, Alexandra; Wal, Jean-Michel; Langella, Philippe

    2003-11-01

    Bovine beta-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice. We constructed a recombinant strain of L. lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L. lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producing L. lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L. lactis strain.

  6. Characterization of a Lactococcus lactis Strain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice

    PubMed Central

    Chatel, Jean-Marc; Nouaille, Sebastien; Adel-Patient, Karine; Le Loir, Yves; Boe, Herman; Gruss, Alexandra; Wal, Jean-Michel; Langella, Philippe

    2003-01-01

    Bovine β-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice. We constructed a recombinant strain of L. lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L. lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producing L. lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L. lactis strain. PMID:14602621

  7. Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action.

    PubMed

    Currie, Richard A; Peffer, Richard C; Goetz, Amber K; Omiecinski, Curtis J; Goodman, Jay I

    2014-07-03

    Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signaling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumors can be viewed as being promoted by a similar PB-like CAR-dependent MOA.

  8. C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

    PubMed Central

    Doroud, Delaram; Zahedifard, Farnaz; Vatanara, Alireza; Taslimi, Yasaman; Vahabpour, Rouholah; Torkashvand, Fatemeh; Vaziri, Behrooz; Rouholamini Najafabadi, Abdolhossein; Rafati, Sima

    2011-01-01

    Background We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average

  9. Functional and Proteomic Investigations Reveal Major Royal Jelly Protein 1 Associated with Anti-hypertension Activity in Mouse Vascular Smooth Muscle Cells

    PubMed Central

    Fan, Pei; Han, Bin; Feng, Mao; Fang, Yu; Zhang, Lan; Hu, Han; Hao, Yue; Qi, Yuping; Zhang, Xiaozhen; Li, Jianke

    2016-01-01

    Vascular smooth muscle cells (VSMCs) are a major cell type of the arterial wall and their functionality is associated with blood pressure regulation. Although royal jelly (RJ) has reported effects on anti-hypertension, the mechanism of blood pressure regulation by major royal jelly protein 1 (MRJP1), the most abundant RJ protein, is still unknown. The mrjp1 gene was inserted into mouse VSMCs to investigate how MRJP1 influences VSMC functionality by functional and proteomic analysis. The expression of MRJP1 in VSMCs significantly reduced cell contraction, migration, and proliferation, suggesting a potential role in decreasing hypertension via action on VSMCs. These anti-hypertension activities were further observed in the changes of the proteome setting of mouse VSMCs. Among 675 different proteins after MRJP1 expression, 646 were down-regulated and significantly enriched in pathways implicated in VSMC contraction and migration, which suggest MRJP1 lowers VSMC contraction and migration by inhibiting muscle filament movement. The down-regulated proteins also enriched pathways in proliferation, indicating that MRJP1 hinders VSMC proliferation by reducing the supply of energy and genetic material. This is the first report integrating MRJP1 into VSMC, revealing the function and mechanism correlated with anti-hypertensive activity. This offers a therapeutic potential to control hypertension by gene-therapy using bee-products. PMID:27444336

  10. Evidence for Msh2 haploinsufficiency in mice revealed by MNU-induced sister-chromatid exchange analysis

    PubMed Central

    Bouffler, S D; Hofland, N; Cox, R; Fodde, R

    2000-01-01

    The role of Msh2 in chromosome stability has been investigated in a targeted mouse model for HNPCC Msh2Δ7N. Chromosome aberration frequencies were similar in bone marrow of Msh2+/+Msh2+/–and Msh2–/–mice and no differential effects of in vivo X-irradiation were noted. By contrast, the induction of sister-chromatid exchanges (SCEs) by methyl nitrosourea (MNU) was reduced in Msh2–/–and Msh2+/–cells to ~20% and ~45% wild-type levels respectively indicating a phenotypic effect of haploinsufficiency of the mouse Msh2 gene. © 2000 CancerResearch Campaign PMID:11044352

  11. Survival analysis of infected mice reveals pathogenic variations in the genome of avian H1N1 viruses.

    PubMed

    Koçer, Zeynep A; Fan, Yiping; Huether, Robert; Obenauer, John; Webby, Richard J; Zhang, Jinghui; Webster, Robert G; Wu, Gang

    2014-12-12

    Most influenza pandemics have been caused by H1N1 viruses of purely or partially avian origin. Here, using Cox proportional hazard model, we attempt to identify the genetic variations in the whole genome of wild-type North American avian H1N1 influenza A viruses that are associated with their virulence in mice by residue variations, host origins of virus (Anseriformes-ducks or Charadriiformes-shorebirds), and host-residue interactions. In addition, through structural modeling, we predicted that several polymorphic sites associated with pathogenicity were located in structurally important sites, especially in the polymerase complex and NS genes. Our study introduces a new approach to identify pathogenic variations in wild-type viruses circulating in the natural reservoirs and ultimately to understand their infectious risks to humans as part of risk assessment efforts towards the emergence of future pandemic strains.

  12. Ebola virus infection kinetics in chimeric mice reveal a key role of T cells as barriers for virus dissemination

    PubMed Central

    Lüdtke, Anja; Ruibal, Paula; Wozniak, David M.; Pallasch, Elisa; Wurr, Stephanie; Bockholt, Sabrina; Gómez-Medina, Sergio; Qiu, Xiangguo; Kobinger, Gary P.; Rodríguez, Estefanía; Günther, Stephan; Krasemann, Susanne; Idoyaga, Juliana; Oestereich, Lisa; Muñoz-Fontela, César

    2017-01-01

    Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome. PMID:28256637

  13. Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.

    PubMed Central

    Hansen, L. A.; Alexander, N.; Hogan, M. E.; Sundberg, J. P.; Dlugosz, A.; Threadgill, D. W.; Magnuson, T.; Yuspa, S. H.

    1997-01-01

    Mice harboring a targeted disruption of the epidermal growth factor receptor (EGFR) allele exhibit a severely disorganized hair follicle phenotype, fuzzy coat, and systemic disease resulting in death before 3 weeks. This skin phenotype was reproduced in whole skin grafts and in grafts of EGFR null hair follicle buds onto nude mice, providing a model to evaluate the natural evolution of skin lacking the EGFR. Hair follicles in grafts of null skin did not progress from anagen to telogen and scanning electron micrografts revealed wavy, flattened hair fibers with cuticular abnormalities. Many of the EGFR null hair follicles in the grafted skin were consumed by an inflammatory reaction resulting in complete hair loss in 67% of the grafts by 10 weeks. Localization of follicular differentiation markers including keratin 6, transglutaminase, and the hair keratins mHa2 and hacl-1 revealed a pattern of premature differentiation within the null hair follicles. In intact EGFR null mice, proliferation in the interfollicular epidermis, but not hair follicles, was greatly decreased in the absence of EGFR. In contrast, grafting of EGFR null skin resulted in a hyperplastic response in the epidermis that did not resolve even after 10 weeks, although the wound-induced hyperplasia in EGFR wild-type grafts had resolved within 3 to 4 weeks. Thus, epithelial expression of the EGFR has complex functions in the skin. It is important in delaying follicular differentiation, may serve to protect the hair follicle from immunological reactions, and modifies both normal and wound-induced epidermal proliferation but seems dispensable for follicular proliferation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9176390

  14. Overexpression of G6PD and HSP90 Beta in Mice with Benzene Exposure Revealed by Serum Peptidome Analysis.

    PubMed

    Zhang, Juan; Tan, Kehong; Meng, Xing; Yang, Wenwen; Wei, Haiyan; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-09-10

    The small peptides representation of the original proteins are a valuable source of information that can be used as biomarkers involved in toxicity mechanism for chemical exposure. The aim of this study is to investigate serum peptide biomarkers of benzene exposure. C57BL/6 mice were enrolled into control group and benzene groups of 150 and 300 mg/kg/d Serum peptides were identified by mass spectrometry using an assisted laser desorption ionization/time of flight mass spectrometry (MS). Differential peptide spectra were obtained by tandem mass spectrometry and analyzed by searching the International Protein Index using the Sequest program. Forty-one peptide peaks were found in the range of 1000-10,000 Da molecular weight. Among them, seven peaks showed significantly different expression between exposure groups and control group. Two peptide peaks (1231.2 and 1241.8), which showed a two-fold increase in expression, were sequenced and confirmed as glucose 6-phosphate dehydrogenase (G6PD) and heat shock protein 90 Beta (HSP90 Beta), respectively. Furthermore, the expression of the two proteins in liver cells showed the same trend as in serum. In conclusion, G6PD and HSP90 beta might be the candidate serum biomarkers of benzene exposure. It also provided possible clues for the molecular mechanism of benzene-induced oxidative stress.

  15. Liver Metabolomics Reveals Increased Oxidative Stress and Fibrogenic Potential in Gfrp Transgenic Mice in Response to Ionizing Radiation

    PubMed Central

    2015-01-01

    Although radiation-induced tissue-specific injury is well documented, the underlying molecular changes resulting in organ dysfunction and the consequences thereof on overall metabolism and physiology have not been elucidated. We previously reported the generation and characterization of a transgenic mouse strain that ubiquitously overexpresses Gfrp (GTPH-1 feedback regulatory protein) and exhibits higher oxidative stress, which is a possible result of decreased tetrahydrobiopterin (BH4) bioavailability. In this study, we report genotype-dependent changes in the metabolic profiles of liver tissue after exposure to nonlethal doses of ionizing radiation. Using a combination of untargeted and targeted quantitative mass spectrometry, we report significant accumulation of metabolites associated with oxidative stress, as well as the dysregulation of lipid metabolism in transgenic mice after radiation exposure. The radiation stress seems to exacerbate lipid peroxidation and also results in higher expression of genes that facilitate liver fibrosis, in a manner that is dependent on the genetic background and post-irradiation time interval. These findings suggest the significance of Gfrp in regulating redox homeostasis in response to stress induced by ionizing radiation affecting overall physiology. PMID:24824572

  16. Liver metabolomics reveals increased oxidative stress and fibrogenic potential in gfrp transgenic mice in response to ionizing radiation.

    PubMed

    Cheema, Amrita K; Pathak, Rupak; Zandkarimi, Fereshteh; Kaur, Prabhjit; Alkhalil, Lynn; Singh, Rajbir; Zhong, Xiaogang; Ghosh, Sanchita; Aykin-Burns, Nukhet; Hauer-Jensen, Martin

    2014-06-06

    Although radiation-induced tissue-specific injury is well documented, the underlying molecular changes resulting in organ dysfunction and the consequences thereof on overall metabolism and physiology have not been elucidated. We previously reported the generation and characterization of a transgenic mouse strain that ubiquitously overexpresses Gfrp (GTPH-1 feedback regulatory protein) and exhibits higher oxidative stress, which is a possible result of decreased tetrahydrobiopterin (BH4) bioavailability. In this study, we report genotype-dependent changes in the metabolic profiles of liver tissue after exposure to nonlethal doses of ionizing radiation. Using a combination of untargeted and targeted quantitative mass spectrometry, we report significant accumulation of metabolites associated with oxidative stress, as well as the dysregulation of lipid metabolism in transgenic mice after radiation exposure. The radiation stress seems to exacerbate lipid peroxidation and also results in higher expression of genes that facilitate liver fibrosis, in a manner that is dependent on the genetic background and post-irradiation time interval. These findings suggest the significance of Gfrp in regulating redox homeostasis in response to stress induced by ionizing radiation affecting overall physiology.

  17. Overexpression of G6PD and HSP90 Beta in Mice with Benzene Exposure Revealed by Serum Peptidome Analysis

    PubMed Central

    Zhang, Juan; Tan, Kehong; Meng, Xing; Yang, Wenwen; Wei, Haiyan; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-01-01

    The small peptides representation of the original proteins are a valuable source of information that can be used as biomarkers involved in toxicity mechanism for chemical exposure. The aim of this study is to investigate serum peptide biomarkers of benzene exposure. C57BL/6 mice were enrolled into control group and benzene groups of 150 and 300 mg/kg/d Serum peptides were identified by mass spectrometry using an assisted laser desorption ionization/time of flight mass spectrometry (MS). Differential peptide spectra were obtained by tandem mass spectrometry and analyzed by searching the International Protein Index using the Sequest program. Forty-one peptide peaks were found in the range of 1000–10,000 Da molecular weight. Among them, seven peaks showed significantly different expression between exposure groups and control group. Two peptide peaks (1231.2 and 1241.8), which showed a two-fold increase in expression, were sequenced and confirmed as glucose 6-phosphate dehydrogenase (G6PD) and heat shock protein 90 Beta (HSP90 Beta), respectively. Furthermore, the expression of the two proteins in liver cells showed the same trend as in serum. In conclusion, G6PD and HSP90 beta might be the candidate serum biomarkers of benzene exposure. It also provided possible clues for the molecular mechanism of benzene-induced oxidative stress. PMID:26378550

  18. Systemic Revealing Pharmacological Signalling Pathway Networks in the Hippocampus of Ischaemia-Reperfusion Mice Treated with Baicalin

    PubMed Central

    Li, Haixia; Zhang, Yingying; Yu, Yanan; Li, Bing; Chen, YinYing; Wu, Hongli; Wang, Jingtao; Li, Jun; Xiong, Xingjiang; He, Qiongyong; Tian, Jinzhou; Wang, Zhong; Wang, Jie

    2013-01-01

    Background. Baicalin (BA) exhibits ill understood neuroprotective, anti-inflammatory, and antioxidative effects in brain injury. Objective. To identify the differential network pathways associated with BA-related biological effects. Methods. MCAO-induced mice received BA 5 mg/Kg (BA group). Controls received vehicle only. Following ischaemia-reperfusion, ArrayTrack analysed the whole genome microarray of hippocampal genes, and MetaCore analysed differentially expressed genes. Results. Four reversing pathways were common to BA and controls, but only 6 were in the top 10 for BA. Three of the top 5 signalling pathways in controls were not observed in BA. BA treatment made absent 3 pathways of the top 5 signalling pathways from the top 5 in controls. There were 2 reversing pathways between controls and BA that showed altered gene expression. Controls had 6 networks associated with cerebral ischaemia. After BA treatment, 9 networks were associated with cerebral ischaemia. Enrichment analysis identified 10 significant biological processes in BA and controls. Of the 10 most significant molecular functions, 7 were common to BA and controls, and only 3 occurred in BA. BA and controls had 7 significant cellular components. Conclusions. This study showed that the clinical effectiveness of BA was based on the complementary effects of multiple pathways and networks. PMID:24381634

  19. Cln1 gene disruption in mice reveals a common pathogenic link between two of the most lethal childhood neurodegenerative lysosomal storage disorders

    PubMed Central

    Chandra, Goutam; Bagh, Maria B.; Peng, Shiyong; Saha, Arjun; Sarkar, Chinmoy; Moralle, Matthew; Zhang, Zhongjian; Mukherjee, Anil B.

    2015-01-01

    Neurodegeneration is a devastating manifestation in the majority of >50 lysosomal storage disorders (LSDs). Neuronal ceroid lipofuscinoses (NCLs) are the most common childhood neurodegenerative LSDs. Mutations in 13 different genes (called CLNs) underlie various types of NCLs, of which the infantile NCL (INCL) and congenital NCL (CNCL) are the most lethal. Although inactivating mutations in the CLN1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) cause INCL, those in the CLN10 gene encoding cathepsin D (CD) underlie CNCL. PPT1 is a lysosomal thioesterase that cleaves the thioester linkage in S-acylated proteins required for their degradation by lysosomal hydrolases like CD. Thus, PPT1 deficiency causes lysosomal accumulation of these lipidated proteins (major constituents of ceroid) leading to INCL. We sought to determine whether there is a common pathogenic link between INCL and CNCL. Using biochemical, histological and confocal microscopic analyses of brain tissues and cells from Cln1−/− mice that mimic INCL, we uncovered that Cln10/CD is overexpressed. Although synthesized in the endoplasmic reticulum, the CD-precursor protein (pro-CD) is transported through endosome to the lysosome where it is proteolytically processed to enzymatically active-CD. We found that despite Cln10 overexpression, the maturation of pro-CD to enzymatically active-CD in lysosome was disrupted. This defect impaired lysosomal degradative function causing accumulation of undegraded cargo in lysosome leading to INCL. Notably, treatment of intact Cln1−/− mice as well as cultured brain cells derived from these animals with a thioesterase-mimetic small molecule, N-tert-butyl-hydroxylamine, ameliorated the CD-processing defect. Our findings are significant in that they define a pathway in which Cln1 mutations disrupt the maturation of a major degradative enzyme in lysosome contributing to neuropathology in INCL and suggest that lysosomal CD deficiency is a common pathogenic link

  20. Major, Trace, and Volatile (CO2, H2O, S, F, and Cl) Elements from 1000+ Hawaiian Olivine-hosted Melt Inclusions Reveal the Dynamics of Crustal Recycling

    NASA Astrophysics Data System (ADS)

    Marske, J. P.; Hauri, E. H.; Trusdell, F.; Garcia, M. O.; Pietruszka, A. J.

    2015-12-01

    Global cycling of volatile elements (H2O, CO2, F, S, Cl) via subduction to deep mantle followed by entrainment and melting within ascending mantle plumes is an enigmatic process that controls key aspects of hot spot volcanism (i.e. melting rate, magma supply, degassing, eruptive style). Variations in radiogenic isotope ratios (e.g.187Os/188Os) at hot spots such as Hawaii reveal magmatic processes within deep-seated mantle plumes (e.g. mantle heterogeneity, lithology, and melt transport). Shield-stage lavas from Hawaii likely originate from a mixed plume source containing peridotite and recycled oceanic crust (pyroxenite) based on variations of radiogenic isotopes. Hawaiian lavas display correlations among isotopes, major and trace elements [1] that might be expected to have an expression in the volatile elements. To investigate this link, we present Os isotopic ratios (n=51), and major, trace, and volatile elements from 1003 olivine-hosted melt inclusions (MI) and their host minerals from tephra from Koolau, Mauna Loa, Hualalai, Kilauea, and Loihi volcanoes. The data show a strong correlation between MI volatile contents and incompatible trace element ratios (La/Yb) with Os isotopes of the same host olivines and reveal large-scale volatile heterogeneity and zonation exists within the Hawaiian plume. 'Loa' chain lavas, which are thought to originate from greater proportions of recycled oceanic crust/pyroxenite, have MIs with lower H2O, S, F, and Cl contents compared to 'Kea' chain lavas that were derived from more peridotite-rich sources. The depletion of volatile elements in the 'Loa' volcano MIs can be explained if they tapped an ancient dehydrated oceanic crust component within the Hawaiian plume. Higher extents of melting beneath 'Loa' volcanoes can also explain these depletions. The presence of dehydrated recycled mafic material in the plume source suggests that subduction effectively devolatilizes part of the oceanic crust. These results are similar to the

  1. Molecular dynamics simulations on pars intercerebralis major peptide-C (PMP-C) reveal the role of glycosylation and disulfide bonds in its enhanced structural stability and function.

    PubMed

    Kaushik, Sandeep; Mohanty, Debasisa; Surolia, Avadhesha

    2012-01-01

    Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29-32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29-32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29-32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.

  2. The Mice at play in the CALIFA survey. A case study of a gas-rich major merger between first passage and coalescence

    NASA Astrophysics Data System (ADS)

    Wild, Vivienne; Rosales-Ortega, Fabian; Falcón-Barroso, Jesus; García-Benito, Rubén; Gallazzi, Anna; González Delgado, Rosa M.; Bekeraité, Simona; Pasquali, Anna; Johansson, Peter H.; García Lorenzo, Begoña; van de Ven, Glenn; Pawlik, Milena; Peréz, Enrique; Monreal-Ibero, Ana; Lyubenova, Mariya; Cid Fernandes, Roberto; Méndez-Abreu, Jairo; Barrera-Ballesteros, Jorge; Kehrig, Carolina; Iglesias-Páramo, Jorge; Bomans, Dominik J.; Márquez, Isabel; Johnson, Benjamin D.; Kennicutt, Robert C.; Husemann, Bernd; Mast, Damian; Sánchez, Sebastian F.; Walcher, C. Jakob; Alves, João; Aguerri, Alfonso L.; Alonso Herrero, Almudena; Bland-Hawthorn, Joss; Catalán-Torrecilla, Cristina; Florido, Estrella; Gomes, Jean Michel; Jahnke, Knud; López-Sánchez, Á. R.; de Lorenzo-Cáceres, Adriana; Marino, Raffaella A.; Mármol-Queraltó, Esther; Olden, Patrick; del Olmo, Ascensión; Papaderos, Polychronis; Quirrenbach, Andreas; Vílchez, Jose M.; Ziegler, Bodo

    2014-07-01

    We present optical integral field spectroscopy (IFS) observations of the Mice, a major merger between two massive (≳1011 M⊙) gas-rich spirals NGC 4676A and B, observed between first passage and final coalescence. The spectra provide stellar and gas kinematics, ionised gas properties, and stellar population diagnostics, over the full optical extent of both galaxies with ~1.6 kpc spatial resolution. The Mice galaxies provide a perfect case study that highlights the importance of IFS data for improving our understanding of local galaxies. The impact of first passage on the kinematics of the stars and gas has been significant, with strong bars most likely induced in both galaxies. The barred spiral NGC 4676B exhibits a strong twist in both its stellar and ionised gas disk. The edge-on disk galaxy NGC 4676A appears to be bulge free, with a strong bar causing its "boxy" light profile. On the other hand, the impact of the merger on the stellar populations has been minimal thus far. By combining the IFS data with archival multiwavelength observations we show that star formation induced by the recent close passage has not contributed significantly to the total star formation rate or stellar mass of the galaxies. Both galaxies show bicones of high ionisation gas extending along their minor axes. In NGC 4676A the high gas velocity dispersion and Seyfert-like line ratios at large scaleheight indicate a powerful outflow. Fast shocks (vs ~ 350 km s-1) extend to ~6.6 kpc above the disk plane. The measured ram pressure (P/k = 4.8 × 106 K cm-3) and mass outflow rate (~8-20 M⊙ yr-1) are similar to superwinds from local ultra-luminous infrared galaxies, although NGC 4676A only has a moderate infrared luminosity of 3 × 1010 L⊙. Energy beyond what is provided by the mechanical energy of the starburst appears to be required to drive the outflow. Finally, we compare the observations to mock kinematic and stellar population maps extracted from a hydrodynamical merger simulation

  3. Metabolomic profiling of urine samples from mice exposed to protons reveals radiation quality and dose specific differences.

    PubMed

    Laiakis, Evagelia C; Trani, Daniela; Moon, Bo-Hyun; Strawn, Steven J; Fornace, Albert J

    2015-04-01

    As space travel is expanding to include private tourism and travel beyond low-Earth orbit, so is the risk of exposure to space radiation. Galactic cosmic rays and solar particle events have the potential to expose space travelers to significant doses of radiation that can lead to increased cancer risk and other adverse health consequences. Metabolomics has the potential to assess an individual's risk by exploring the metabolic perturbations in a biofluid or tissue. In this study, C57BL/6 mice were exposed to 0.5 and 2 Gy of 1 GeV/nucleon of protons and the levels of metabolites were evaluated in urine at 4 h after radiation exposure through liquid chromatography coupled to time-of-flight mass spectrometry. Significant differences were identified in metabolites that map to the tricarboxylic acid (TCA) cycle and fatty acid metabolism, suggesting that energy metabolism is severely impacted after exposure to protons. Additionally, various pathways of amino acid metabolism (tryptophan, tyrosine, arginine and proline and phenylalanine) were affected with potential implications for DNA damage repair and cognitive impairment. Finally, presence of products of purine and pyrimidine metabolism points to direct DNA damage or increased apoptosis. Comparison of these metabolomic data to previously published data from our laboratory with gamma radiation strongly suggests a more pronounced effect on metabolism with protons. This is the first metabolomics study with space radiation in an easily accessible biofluid such as urine that further investigates and exemplifies the biological differences at early time points after exposure to different radiation qualities.

  4. Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice

    PubMed Central

    Swanson, Kelly S; Dowd, Scot E; Suchodolski, Jan S; Middelbos, Ingmar S; Vester, Brittany M; Barry, Kathleen A; Nelson, Karen E; Torralba, Manolito; Henrissat, Bernard; Coutinho, Pedro M; Cann, Isaac KO; White, Bryan A; Fahey, George C

    2011-01-01

    This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503 280 (K9C) and 505 061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease. PMID:20962874

  5. An allelic series of mice reveals a role for RERE in the development of multiple organs affected in chromosome 1p36 deletions.

    PubMed

    Kim, Bum Jun; Zaveri, Hitisha P; Shchelochkov, Oleg A; Yu, Zhiyin; Hernández-García, Andrés; Seymour, Michelle L; Oghalai, John S; Pereira, Fred A; Stockton, David W; Justice, Monica J; Lee, Brendan; Scott, Daryl A

    2013-01-01

    Individuals with terminal and interstitial deletions of chromosome 1p36 have a spectrum of defects that includes eye anomalies, postnatal growth deficiency, structural brain anomalies, seizures, cognitive impairment, delayed motor development, behavior problems, hearing loss, cardiovascular malformations, cardiomyopathy, and renal anomalies. The proximal 1p36 genes that contribute to these defects have not been clearly delineated. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in this region and encodes a nuclear receptor coregulator that plays a critical role in embryonic development as a positive regulator of retinoic acid signaling. Rere-null mice die of cardiac failure between E9.5 and E11.5. This limits their usefulness in studying the role of RERE in the latter stages of development and into adulthood. To overcome this limitation, we created an allelic series of RERE-deficient mice using an Rere-null allele, om, and a novel hypomorphic Rere allele, eyes3 (c.578T>C, p.Val193Ala), which we identified in an N-ethyl-N-nitrosourea (ENU)-based screen for autosomal recessive phenotypes. Analyses of these mice revealed microphthalmia, postnatal growth deficiency, brain hypoplasia, decreased numbers of neuronal nuclear antigen (NeuN)-positive hippocampal neurons, hearing loss, cardiovascular malformations-aortic arch anomalies, double outlet right ventricle, and transposition of the great arteries, and perimembranous ventricular septal defects-spontaneous development of cardiac fibrosis and renal agenesis. These findings suggest that RERE plays a critical role in the development and function of multiple organs including the eye, brain, inner ear, heart and kidney. It follows that haploinsufficiency of RERE may contribute-alone or in conjunction with other genetic, environmental, or stochastic factors-to the development of many of the phenotypes seen in individuals with terminal and interstitial deletions that include the proximal region of

  6. Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice*

    PubMed Central

    Schwindinger, William F.; Mirshahi, Uyenlinh L.; Baylor, Kelly A.; Sheridan, Kathleen M.; Stauffer, Anna M.; Usefof, Stephanie; Stecker, Mark M.; Mirshahi, Tooraj; Robishaw, Janet D.

    2012-01-01

    The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ3 and γ7 subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3−/−Gng7−/− double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ3 subunit is a component of a Gi/o protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ7 subunit is a component of a Golf protein that is responsible for A2A adenosine or D1 dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process. PMID:22207761

  7. Comparative sequence analysis of the potato cyst nematode resistance locus H1 reveals a major lack of co-linearity between three haplotypes in potato (Solanum tuberosum ssp.).

    PubMed

    Finkers-Tomczak, Anna; Bakker, Erin; de Boer, Jan; van der Vossen, Edwin; Achenbach, Ute; Golas, Tomasz; Suryaningrat, Suwardi; Smant, Geert; Bakker, Jaap; Goverse, Aska

    2011-02-01

    The H1 locus confers resistance to the potato cyst nematode Globodera rostochiensis pathotypes 1 and 4. It is positioned at the distal end of chromosome V of the diploid Solanum tuberosum genotype SH83-92-488 (SH) on an introgression segment derived from S. tuberosum ssp. andigena. Markers from a high-resolution genetic map of the H1 locus (Bakker et al. in Theor Appl Genet 109:146-152, 2004) were used to screen a BAC library to construct a physical map covering a 341-kb region of the resistant haplotype coming from SH. For comparison, physical maps were also generated of the two haplotypes from the diploid susceptible genotype RH89-039-16 (S. tuberosum ssp. tuberosum/S. phureja), spanning syntenic regions of 700 and 319 kb. Gene predictions on the genomic segments resulted in the identification of a large cluster consisting of variable numbers of the CC-NB-LRR type of R genes for each haplotype. Furthermore, the regions were interspersed with numerous transposable elements and genes coding for an extensin-like protein and an amino acid transporter. Comparative analysis revealed a major lack of gene order conservation in the sequences of the three closely related haplotypes. Our data provide insight in the evolutionary mechanisms shaping the H1 locus and will facilitate the map-based cloning of the H1 resistance gene.

  8. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface.

    PubMed

    Atkison, James H; Parnham, Stuart; Marcotte, William R; Olsen, Shaun K

    2016-09-02

    Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct.

  9. Linkage and regional association analysis reveal two new tightly-linked major-QTLs for pod number and seed number per pod in rapeseed (Brassica napus L.).

    PubMed

    Shi, Jiaqin; Zhan, Jiepeng; Yang, Yuhua; Ye, Jiang; Huang, Shunmou; Li, Ruiyuan; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

    2015-10-05

    To facilitate the pseudochromosomes assembly and gene cloning in rapeseed, we developed a reference genetic population/map (named BnaZNF2) from two sequenced cultivars, Zhongshuang11 and No.73290, those exhibit significant differences in many traits, particularly yield components. The BnaZNF2 genetic map exhibited perfect collinearity with the physical map of B. napus, indicating its high quality. Comparative mapping revealed several genomic rearrangements between B. napus and B. rapa or B. oleracea. A total of eight and 16 QTLs were identified for pod number and seed number per pod, respectively, and of which three and five QTLs are identical to previously identified ones, whereas the other five and 11 are novel. Two new major QTL respectively for pod number and seed number per pod, qPN.A06-1 and qSN.A06-1 (R(2 )= 22.8% and 32.1%), were colocalised with opposite effects, and only qPN.A06-1 was confirmed and narrowed by regional association analysis to 180 kb including only 33 annotated genes. Conditional QTL analysis and subsequent NILs test indicated that tight linkage, rather than pleiotropy, was the genetic causation of their colocalisation. Our study demonstrates potential of this reference genetic population/map for precise QTL mapping and as a base for positional gene cloning in rapeseed.

  10. Linkage and regional association analysis reveal two new tightly-linked major-QTLs for pod number and seed number per pod in rapeseed (Brassica napus L.)

    PubMed Central

    Shi, Jiaqin; Zhan, Jiepeng; Yang, Yuhua; Ye, Jiang; Huang, Shunmou; Li, Ruiyuan; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

    2015-01-01

    To facilitate the pseudochromosomes assembly and gene cloning in rapeseed, we developed a reference genetic population/map (named BnaZNF2) from two sequenced cultivars, Zhongshuang11 and No.73290, those exhibit significant differences in many traits, particularly yield components. The BnaZNF2 genetic map exhibited perfect collinearity with the physical map of B. napus, indicating its high quality. Comparative mapping revealed several genomic rearrangements between B. napus and B. rapa or B. oleracea. A total of eight and 16 QTLs were identified for pod number and seed number per pod, respectively, and of which three and five QTLs are identical to previously identified ones, whereas the other five and 11 are novel. Two new major QTL respectively for pod number and seed number per pod, qPN.A06-1 and qSN.A06-1 (R2 = 22.8% and 32.1%), were colocalised with opposite effects, and only qPN.A06-1 was confirmed and narrowed by regional association analysis to 180 kb including only 33 annotated genes. Conditional QTL analysis and subsequent NILs test indicated that tight linkage, rather than pleiotropy, was the genetic causation of their colocalisation. Our study demonstrates potential of this reference genetic population/map for precise QTL mapping and as a base for positional gene cloning in rapeseed. PMID:26434411

  11. Gene targeting study reveals unexpected expression of brain-expressed X-linked 2 in endocrine and tissue stem/progenitor cells in mice.

    PubMed

    Ito, Keiichi; Yamazaki, Satoshi; Yamamoto, Ryo; Tajima, Yoko; Yanagida, Ayaka; Kobayashi, Toshihiro; Kato-Itoh, Megumi; Kakuta, Shigeru; Iwakura, Yoichiro; Nakauchi, Hiromitsu; Kamiya, Akihide

    2014-10-24

    Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.

  12. Susceptibility of Inbred Mice to Leishmania major Infection: Genetic Analysis of Macrophage Activation and Innate Resistance to Disease in Individual Progeny of P/J (Susceptible) and C3H/HeN (Resistant) Mice

    DTIC Science & Technology

    1990-12-01

    mediated immu- ease and defective macrophage activation in Bx mice that nity in mice highly susceptible to Leishmania tropica . J. Exp. could not be...inbred mice to Leishmania tropica infec- tion: correlation of susceptibility with in vitro defective macro- LITERATURE CITED phage microbicidal...probability and phage activation to kill Leishmania tropica : characterization of statistics. Chemical Rubber Co., Cleveland. P/J mouse macrophage defects for

  13. Green tea and its major components ameliorate immune dysfunction in mice bearing Lewis lung carcinoma and treated with the carcinogen NNK.

    PubMed

    Zhu, M; Gong, Y; Yang, Z; Ge, G; Han, C; Chen, J

    1999-01-01

    The protective effects of tea and/or its components on dysfunction of immune functions during tumor growth and carcinogenesis in mice were studied using two experimental models: C57/BL6J mice transplanted with Lewis lung carcinoma (LLC) and Kunming mice treated with a single dose of 4-(methylnitrosamino-)-1-(3-pyridyl)-1-butanone (NNK). In C57/BL6J mice bearing LLC, the weight of the thymus decreased, the proportion of CD4(+)-positive T lymphocytes and the ratio of CD4+ to CD8+ decreased, luminol-enhanced chemiluminescence of white blood cells in peripheral blood stimulated by zymosan increased, and plaque-forming cells (PFC) decreased. However, in LLC-bearing mice given green tea as drinking water, all immune functions were improved, along with inhibition of tumor growth. In Kunming mice treated with NNK, during the four weeks of observation, their immunologic indicators, such as phagocytosis of macrophages in the abdominal cavity, luminol-enhanced chemiluminescence of white blood cells, plaque-forming cells, and delayed-type hypersensitivity, increased or decreased to various extents compared with normal controls. However, these changes were significantly prevented in the mice given green tea, mixed tea, or tea polyphenol as drinking water. In conclusion, tea and its components ameliorated immune dysfunction in mice bearing LLC or treated with the carcinogen NNK.

  14. Proteomic Analysis of Tissue from α1,3-galactosyltransferase Knockout Mice Reveals That a Wide Variety of Proteins and Protein Fragments Change Expression Level

    PubMed Central

    Thorlacius-Ussing, Louise; Ludvigsen, Maja; Kirkeby, Svend

    2013-01-01

    A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope) expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient’s blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography - tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes. PMID:24244699

  15. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

    PubMed Central

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys. PMID:27010944

  16. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

    PubMed

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

  17. Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome

    PubMed Central

    2013-01-01

    Background Listeria monocytogenes is an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in the Galleria mellonella insect model. Results The species pan-genome of L. monocytogenes is highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like), and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genus Listeria imply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of species L. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates supports observations of a

  18. Microarray Analysis Reveals Increased Transcriptional Repression and Reduced Metabolic Activity but Not Major Changes in the Core Apoptotic Machinery during Maturation of Sympathetic Neurons

    PubMed Central

    Raba, Mikk; Palgi, Jaan; Lehtivaara, Maria; Arumäe, Urmas

    2016-01-01

    Postnatal maturation of the neurons whose main phenotype and basic synaptic contacts are already established includes neuronal growth, refinement of synaptic contacts, final steps of differentiation, programmed cell death period (PCD) etc. In the sympathetic neurons, postnatal maturation includes permanent end of the PCD that occurs with the same time schedule in vivo and in vitro suggesting that the process could be genetically determined. Also many other changes in the neuronal maturation could be permanent and thus based on stable changes in the genome expression. However, postnatal maturation of the neurons is poorly studied. Here we compared the gene expression profiles of immature and mature sympathetic neurons using Affymetrix microarray assay. We found 1310 significantly up-regulated and 1151 significantly down-regulated genes in the mature neurons. Gene ontology analysis reveals up-regulation of genes related to neuronal differentiation, chromatin and epigenetic changes, extracellular factors and their receptors, and cell adhesion, whereas many down-regulated genes were related to metabolic and biosynthetic processes. We show that termination of PCD is not related to major changes in the expression of classical genes for apoptosis or cell survival. Our dataset is deposited to the ArrayExpress database and is a valuable source to select candidate genes in the studies of neuronal maturation. As an example, we studied the changes in the expression of selected genes Igf2bp3, Coro1A, Zfp57, Dcx, and Apaf1 in the young and mature sympathetic ganglia by quantitative PCR and show that these were strongly downregulated in the mature ganglia. PMID:27013977

  19. Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice.

    PubMed

    Miyashita, Takenori; Burford, James L; Hong, Young-Kwon; Gevorgyan, Haykanush; Lam, Lisa; Hoshikawa, Hiroshi; Mori, Nozomu; Peti-Peterdi, Janos

    2015-01-30

    This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

  20. An Important Role for Major Histocompatibility Complex Class I-Restricted T Cells, and a Limited Role for Gamma Interferon, in Protection of Mice against Lethal Herpes Simplex Virus Infection

    PubMed Central

    Holterman, Ai-Xuan; Rogers, Kathleen; Edelmann, Kurt; Koelle, David M.; Corey, Lawrence; Wilson, Christopher B.

    1999-01-01

    Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-γ) has been proposed. To address the roles of MHC class I and of IFN-γ, C57BL/6 mice deficient in MHC class I expression (β2 microglobulin knockout [β2KO] mice), in IFN-γ expression (IFN-γKO mice), or in both (IFN-γKO/β2KO mice) were infected with HSV by footpad inoculation. β2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-γ appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-γ. PMID:9971787

  1. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain.

    PubMed

    Nassar, Mohammed A; Stirling, L Caroline; Forlani, Greta; Baker, Mark D; Matthews, Elizabeth A; Dickenson, Anthony H; Wood, John N

    2004-08-24

    Nine voltage-gated sodium channels are expressed in complex patterns in mammalian nerve and muscle. Three channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are expressed selectively in peripheral damage-sensing neurons. Because there are no selective blockers of these channels, we used gene ablation in mice to examine the function of Na(v)1.7 (PN1) in pain pathways. A global Na(v)1.7-null mutant was found to die shortly after birth. We therefore used the Cre-loxP system to generate nociceptor-specific knockouts. Na(v)1.8 is only expressed in peripheral, mainly nociceptive, sensory neurons. We knocked Cre recombinase into the Na(v)1.8 locus to generate heterozygous mice expressing Cre recombinase in Na(v)1.8-positive sensory neurons. Crossing these animals with mice where Na(v)1.7 exons 14 and 15 were flanked by loxP sites produced nociceptor-specific knockout mice that were viable and apparently normal. These animals showed increased mechanical and thermal pain thresholds. Remarkably, all inflammatory pain responses evoked by a range of stimuli, such as formalin, carrageenan, complete Freund's adjuvant, or nerve growth factor, were reduced or abolished. A congenital pain syndrome in humans recently has been mapped to the Na(v)1.7 gene, SCN9A. Dominant Na(v)1.7 mutations lead to edema, redness, warmth, and bilateral pain in human erythermalgia patients, confirming an important role for Na(v)1.7 in inflammatory pain. Nociceptor-specific gene ablation should prove useful in understanding the role of other broadly expressed genes in pain pathways.

  2. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain

    PubMed Central

    Nassar, Mohammed A.; Stirling, L. Caroline; Forlani, Greta; Baker, Mark D.; Matthews, Elizabeth A.; Dickenson, Anthony H.; Wood, John N.

    2004-01-01

    Nine voltage-gated sodium channels are expressed in complex patterns in mammalian nerve and muscle. Three channels, Nav1.7, Nav1.8, and Nav1.9, are expressed selectively in peripheral damage-sensing neurons. Because there are no selective blockers of these channels, we used gene ablation in mice to examine the function of Nav1.7 (PN1) in pain pathways. A global Nav1.7-null mutant was found to die shortly after birth. We therefore used the Cre-loxP system to generate nociceptor-specific knockouts. Nav1.8 is only expressed in peripheral, mainly nociceptive, sensory neurons. We knocked Cre recombinase into the Nav1.8 locus to generate heterozygous mice expressing Cre recombinase in Nav1.8-positive sensory neurons. Crossing these animals with mice where Nav1.7 exons 14 and 15 were flanked by loxP sites produced nociceptor-specific knockout mice that were viable and apparently normal. These animals showed increased mechanical and thermal pain thresholds. Remarkably, all inflammatory pain responses evoked by a range of stimuli, such as formalin, carrageenan, complete Freund's adjuvant, or nerve growth factor, were reduced or abolished. A congenital pain syndrome in humans recently has been mapped to the Nav1.7 gene, SCN9A. Dominant Nav1.7 mutations lead to edema, redness, warmth, and bilateral pain in human erythermalgia patients, confirming an important role for Nav1.7 in inflammatory pain. Nociceptor-specific gene ablation should prove useful in understanding the role of other broadly expressed genes in pain pathways. PMID:15314237

  3. Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

    PubMed

    Dupuis, L; de Tapia, M; René, F; Lutz-Bucher, B; Gordon, J W; Mercken, L; Pradier, L; Loeffler, J P

    2000-08-01

    In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

  4. Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice.

    PubMed

    Majer, Anna; Medina, Sarah J; Niu, Yulian; Abrenica, Bernard; Manguiat, Kathy J; Frost, Kathy L; Philipson, Clark S; Sorensen, Debra L; Booth, Stephanie A

    2012-01-01

    Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. We found that a major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response that is mediated, at least in part, by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in

  5. Analysis of YFP(J16)-R6/2 reporter mice and postmortem brains reveals early pathology and increased vulnerability of callosal axons in Huntington's disease.

    PubMed

    Gatto, Rodolfo G; Chu, Yaping; Ye, Allen Q; Price, Steven D; Tavassoli, Ehsan; Buenaventura, Andrea; Brady, Scott T; Magin, Richard L; Kordower, Jeffrey H; Morfini, Gerardo A

    2015-09-15

    Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.

  6. Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane.

    PubMed

    Legan, P Kevin; Goodyear, Richard J; Morín, Matías; Mencia, Angeles; Pollard, Hilary; Olavarrieta, Leticia; Korchagina, Julia; Modamio-Hoybjor, Silvia; Mayo, Fernando; Moreno, Felipe; Moreno-Pelayo, Miguel-Angel; Richardson, Guy P

    2014-05-15

    Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype-phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the Tecta(L1820F,G1824D/+) mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the Tecta(C1837G/+) mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the Tecta(C1619S/+) mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8-40 kHz range are elevated by 30-40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20-30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

  7. Surfactant protein-C chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells.

    PubMed

    Lee, Joo-Hyeon; Kim, Jonghwan; Gludish, David; Roach, Rebecca R; Saunders, Arven H; Barrios, Juliana; Woo, Andrew Jonghan; Chen, Huaiyong; Conner, David A; Fujiwara, Yuko; Stripp, Barry R; Kim, Carla F

    2013-03-01

    The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.

  8. Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: Investigation of nicotinic acid receptor agonists

    SciTech Connect

    Zgoda-Pols, Joanna R.; Chowdhury, Swapan; Wirth, Mark; Milburn, Michael V.; Alexander, Danny C.; Alton, Kevin B.

    2011-08-15

    An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea. An LC-MS assay for quantitative determination of 3IS in mouse matrices was also developed. Following treatment with SCH 900424, 3IS levels were markedly increased in murine plasma and brain, thereby potentially contributing to renal- and central nervous system (CNS)-related rapid onset of toxicities. Furthermore, significant decrease in urinary excretion of 3IS in those animals due to compromised renal function may be associated with the elevation of 3IS in plasma and brain. These data suggest that 3IS has a potential to be a marker of renal and CNS toxicities during chemically-induced AKI in mice. In addition, based on the metabolomic analysis other statistically significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurenate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. This comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development. - Research Highlights: > Nicotinic acid receptor agonist, SCH 900424, caused acute kidney injury in mice. > MS-based metabolomics was conducted to identify potential small molecule markers of renal toxicity. > 3-indoxyl-sulfate was found to be as a more sensitive marker of renal toxicity than creatinine

  9. Eumelanin is a major determinant for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) incorporation into hair of mice.

    PubMed

    Hegstad, Soffrid; Reistad, Ragnhild; Haug, Line S; Alexander, Jan

    2002-06-01

    Mice with different hair pigmentation were studied to evaluate the role of melanin in the incorporation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair. Mice C57BL/6J-c2j/+ (white), C57BL/6J-Ay (yellow), C57L/J (grey), C57BR/cdJ (brown) and C57BL/6J (black) were dosed with PhIP: 7-9 days old (total amount: 0.006 or 0.58 mg/kg b.wt., for 4 days) and adults (total amount 50 mg/kg b.wt. during 8 weeks). Hair was collected either 30 days after the last PhIP administration (new-born mice) or 8 weeks after the first administration (adult mice). PhIP was incorporated into black hair to a greater extent than into brown, grey, yellow and non-pigmented hair. The concentration of PhIP in the hair of new-born mice exposed to 0.58 mg PhIP/kg b.wt. were (mean+/-S.D.): 328+/-135 (black), 134+/-41 (brown), 9.1+/-1.2 (yellow) and 5.2+/-1.4 (white) ng/g hair. The PhIP concentrations in the hair of adult mice exposed to 50 mg/kg b.wt. were: 4750+/-1449 (black), 810+/-235 (brown), 541+/-119 (grey), 35.5+/-4.6 (yellow) and 21.6+/-8.8 (white) ng/g, and the eumelanin hair concentration in the same animals decreased in a similar pattern. A linear relationship (r2= 1.00, P<0.0001) between the relative PhIP incorporation and the eumelanin concentration in hair was found.

  10. The Major Determinant of Attenuation in Mice of the Candid1 Vaccine for Argentine Hemorrhagic Fever Is Located in the G2 Glycoprotein Transmembrane Domain▿

    PubMed Central

    Albariño, César G.; Bird, Brian H.; Chakrabarti, Ayan K.; Dodd, Kimberly A.; Flint, Mike; Bergeron, Éric; White, David M.; Nichol, Stuart T.

    2011-01-01

    Candid1, a live-attenuated Junin virus vaccine strain, was developed during the early 1980s to control Argentine hemorrhagic fever, a severe and frequently fatal human disease. Six amino acid substitutions were found to be unique to this vaccine strain, and their role in virulence attenuation in mice was analyzed using a series of recombinant viruses. Our results indicate that Candid1 is attenuated in mice through a single amino acid substitution in the transmembrane domain of the G2 glycoprotein. This work provides insight into the molecular mechanisms of attenuation of the only arenavirus vaccine currently available. PMID:21795336

  11. Analysis of mice deficient in both REV1 catalytic activity and POLH reveals an unexpected role for POLH in the generation of C to G and G to C transversions during Ig gene hypermutation.

    PubMed

    Kano, Chie; Hanaoka, Fumio; Wang, Ji-Yang

    2012-03-01

    Multiple DNA polymerases are involved in the generation of somatic mutations during Ig gene hypermutation. Mice expressing a catalytically inactive REV1 (REV1AA) exhibit reduction of both C to G and G to C transversions and moderate decrease of A/T mutations, whereas DNA polymerase η (POLH) deficiency causes greatly reduced A/T mutations. To investigate whether REV1 and POLH interact genetically and functionally during Ig gene hypermutation, we established REV1AA Polh(-/-) mice and analyzed Ig gene hypermutation in the germinal center (GC) B cells. REV1AA Polh(-/-) mice were born at the expected ratio and developed normally with no apparent gross abnormalities. B-cell development, maturation, Ig gene class switch and the GC B-cell expansion were not affected in these mice. REV1AA Polh(-/-) B cells also exhibited relatively normal sensitivity to etoposide and ionizing radiation. Analysis of somatic mutations in the J(H)4 intronic region revealed that REV1AA Polh(-/-) mice had a further decrease of overall mutation frequency compared with REV1AA or Polh(-/-) mice, indicating that the double deficiency additively affected the generation of mutations. Remarkably, REV1AA Polh(-/-) mice had nearly absent C to G and G to C transversions, suggesting that POLH is essential for the generation of residual C to G and G to C transversions observed in REV1AA mice. These results reveal genetic interactions between REV1 catalytic activity and POLH and identify an alternative pathway, mediated by non-catalytic REV1 and POLH, in the generation of C to G and G to C transversions.

  12. Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil.

    PubMed

    Tsuboyama-Kasaoka, Nobuyo; Sano, Kayo; Shozawa, Chikako; Osaka, Toshimasa; Ezaki, Osamu

    2008-03-01

    Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

  13. Of mice and the 'Age of Discovery': the complex history of colonization of the Azorean archipelago by the house mouse (Mus musculus) as revealed by mitochondrial DNA variation.

    PubMed

    Gabriel, S I; Mathias, M L; Searle, J B

    2015-01-01

    Humans have introduced many species onto remote oceanic islands. The house mouse (Mus musculus) is a human commensal and has consequently been transported to oceanic islands around the globe as an accidental stowaway. The history of these introductions can tell us not only about the mice themselves but also about the people that transported them. Following a phylogeographic approach, we used mitochondrial D-loop sequence variation (within an 849- to 864-bp fragment) to study house mouse colonization of the Azores. A total of 239 sequences were obtained from all nine islands, and interpretation was helped by previously published Iberian sequences and 66 newly generated Spanish sequences. A Bayesian analysis revealed presence in the Azores of most of the D-loop clades previously described in the domesticus subspecies of the house mouse, suggesting a complex colonization history of the archipelago as a whole from multiple geographical origins, but much less heterogeneity (often single colonization?) within islands. The expected historical link with mainland Portugal was reflected in the pattern of D-loop variation of some of the islands but not all. A more unexpected association with a distant North European source area was also detected in three islands, possibly reflecting human contact with the Azores prior to the 15th century discovery by Portuguese mariners. Widening the scope to colonization of the Macaronesian islands as a whole, human linkages between the Azores, Madeira, the Canaries, Portugal and Spain were revealed through the sharing of mouse sequences between these areas. From these and other data, we suggest mouse studies may help resolve historical uncertainties relating to the 'Age of Discovery'.

  14. Potentiation of the bioavailability of blueberry phenolic compounds by co-ingested grape phenolic compounds in mice, revealed by targeted metabolomic profiling in plasma and feces.

    PubMed

    Dudonné, Stéphanie; Dal-Pan, Alexandre; Dubé, Pascal; Varin, Thibault V; Calon, Frédéric; Desjardins, Yves

    2016-08-10

    The low bioavailability of dietary phenolic compounds, resulting from poor absorption and high rates of metabolism and excretion, is a concern as it can limit their potential beneficial effects on health. Targeted metabolomic profiling in plasma and feces of mice supplemented for 15 days with a blueberry extract, a grape extract or their combination revealed significantly increased plasma concentrations (3-5 fold) of blueberry phenolic metabolites in the presence of a co-ingested grape extract, associated with an equivalent decrease in their appearance in feces. Additionally, the repeated daily administration of the blueberry-grape combination significantly increased plasma phenolic concentrations (2-3-fold) compared to animals receiving only a single acute dose, with no such increase being observed with individual extracts. These findings highlight a positive interaction between blueberry and grape constituents, in which the grape extract enhanced the absorption of blueberry phenolic compounds. This study provides for the first time in vivo evidence of such an interaction occurring between co-ingested phenolic compounds from fruit extracts leading to their improved bioavailability.

  15. Microarray Analysis Reveals Higher Gestational Folic Acid Alters Expression of Genes in the Cerebellum of Mice Offspring—A Pilot Study

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Chadman, Kathryn K.; Brown, W. Ted; Junaid, Mohammed A.

    2015-01-01

    Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development. PMID:25629700

  16. Preserved energy balance in mice lacking FoxO1 in neurons of Nkx2.1 lineage reveals functional heterogeneity of FoxO1 signaling within the hypothalamus.

    PubMed

    Heinrich, Garrett; Meece, Kana; Wardlaw, Sharon L; Accili, Domenico

    2014-05-01

    Transcription factor forkhead box O1 (FoxO1) regulates energy expenditure (EE), food intake, and hepatic glucose production. These activities have been mapped to specific hypothalamic neuronal populations using cell type-specific knockout experiments in mice. To parse out the integrated output of FoxO1-dependent transcription from different neuronal populations and multiple hypothalamic regions, we used transgenic mice expressing Cre recombinase from the Nkx2.1 promoter to ablate loxP-flanked Foxo1 alleles from a majority of hypothalamic neurons (Foxo1KO(Nkx2.1) mice). This strategy resulted in the expected inhibition of FoxO1 expression, but only produced a transient reduction of body weight as well as a decreased body length. The transient decrease of body weight in male mice was accompanied by decreased fat mass. Male Foxo1KO(Nkx2.1) mice show food intake similar to that in wild-type controls, and, although female knockout mice eat less, they do so in proportion to a reduced body size. EE is unaffected in Foxo1KO(Nkx2.1) mice, although small increases in body temperature are present. Unlike other neuron-specific Foxo1 knockout mice, Foxo1KO(Nkx2.1) mice are not protected from diet-induced obesity. These studies indicate that, unlike the metabolic effects of highly restricted neuronal subsets (proopiomelanocortin, neuropeptide Y/agouti-related peptide, and steroidogenic factor 1), those of neurons derived from the Nkx2.1 lineage either occur in a FoxO1-independent fashion or are compensated for through developmental plasticity.

  17. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  18. Enriching the Environment of [alpha]CaMKII[superscript T286A] Mutant Mice Reveals that LTD Occurs in Memory Processing but Must be Subsequently Reversed by LTP

    ERIC Educational Resources Information Center

    Soto, Florentina; Giese, K. Peter; Edwards, Frances A.; Parsley, Stephanie L.; Pilgram, Sara M.

    2007-01-01

    [alpha]CaMKII[superscript T286A] mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of [alpha]CaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate…

  19. Effect of Polyvinyl Alcohol (PVA) Containing Artemether in Treatment of Cutaneous Leishmaniasis Caused by Leishmania major in BALB/c Mice

    PubMed Central

    Ebrahimisadr, Parisa; Ghaffarifar, Fatemeh; Hassan, Zuhir Mohammad; Sirousazar, Mohammad; Mohammadnejad, Fatemeh

    2014-01-01

    Background: Polyvinyl alcohol (PVA) is one of the well-known polymers, which has been used in numerous biomedical applications because of its good biocompatibility. Objectives: Due to problems made by the therapeutics already used for leishmaniasis, the aim of this study was to evaluate the effect of PVA containing artemether in treating cutaneous leishmaniasis in BALB/c mice. Materials and Methods: Aqueous solution of PVA was prepared by mixing with Double Distilled Water. After preparation of PVA, 4.33 mg of each drug (main drug artemether and control drug 14% glucantime) was added to 100 g of prepared PVA-honey solution. The solution was incubated at 37°C and the release of artemether was evaluated by measuring absorbance at 260 nm wave length. In this study for treatment of mice lesion, we used PVA containing artemether and glucantime and this method was compared with ointment treatment. Results: Mean diameters of lesions in mice treated with artemether were smaller than the control group and the differences were significant (P < 0.05). The mean lesion size of mice treated with PVA containing artemether in comparison with the group treated with ointment of artemether were smaller and the differences were significant (P < 0.05). Conclusions: PVA containing artemether is a new method for treatment of cutaneous leishmaniasis and according to the obtained results, artemether is an appropriate and effective drug, especially when used with PVA as a lesion dressing; thus we suggest that this method can be applied for the treatment of cutaneous leishmaniasis. PMID:25147717

  20. Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice* ♦

    PubMed Central

    Shelkovnikova, Tatyana A.; Peters, Owen M.; Deykin, Alexey V.; Connor-Robson, Natalie; Robinson, Hannah; Ustyugov, Alexey A.; Bachurin, Sergey O.; Ermolkevich, Tatyana G.; Goldman, Igor L.; Sadchikova, Elena R.; Kovrazhkina, Elena A.; Skvortsova, Veronica I.; Ling, Shuo-Chien; Da Cruz, Sandrine; Parone, Philippe A.; Buchman, Vladimir L.; Ninkina, Natalia N.

    2013-01-01

    Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice. PMID:23867462

  1. Effects of a major androgen-dependent urinary protein,. alpha. 2u-globulin on the pituitary-gonadal axis and hypothalamic monoamines in adult male mice

    SciTech Connect

    Ghosh, P.K.; Chandrashekar, V.; Steger, R. Bartke, A. )

    1990-01-01

    The purpose of the present study was to evaluate the effects of alpha-2u-globulin, a sex-dependent male rat urinary protein on pituitary-gonadal functions and hypothalamic monamine contents in male mice. Adult male mice, maintained under standardized laboratory conditions were injected subcutaneously with alpha-2u-globulin or with vehicle daily for 14 days and killed 16 h after the last injection. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and testicular levels of T were measured by radioimmunoassays. The concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in medial basal hypothalamus (MBH) and anterior hypothalamus (AH) were measured by high performance liquid chromatography. Administration of alpha-2u-globulin led to a significant increase in plasma FSH and LH levels. In the MBH of alpha-2u-globulin treated mice, there were significant elevations of NE, DA and 5-HT contents. In the AH, both DA and 5-HT contents were decreased while NE content remained unaltered.

  2. Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

    PubMed

    Waldburger, J M; Suter, T; Fontana, A; Acha-Orbea, H; Reith, W

    2001-08-20

    MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

  3. Systemic administration of 2-hydroxypropyl-β-cyclodextrin to symptomatic Npc1-deficient mice slows cholesterol sequestration in the major organs and improves liver function.

    PubMed

    Lopez, Adam M; Terpack, Sandi J; Posey, Kenneth S; Liu, Benny; Ramirez, Charina M; Turley, Stephen D

    2014-10-01

    In Niemann-Pick type C (NPC) disease, loss-of-function mutations in either NPC1 or NPC2 result in progressive accumulation of unesterified cholesterol (UC) and glycosphingolipids in all organs, leading to neurodegeneration, pulmonary dysfunction and sometimes liver failure. There is no cure for this disorder. Studies using primarily NPC mouse models have shown that systemic administration of 2-hydroxypropyl-β-cyclodextrin (2HPβCD), starting in early neonatal life, diminishes UC accumulation in most organs, slows disease progression and extends lifespan. The key question now is whether delaying the start of 2HPβCD treatment until early adulthood, when the amount of entrapped UC throughout the body is markedly elevated, has any of the benefits found when treatment begins at 7 days of age. In the present study, Npc1(-/-) and Npc1(+/+) mice were given saline or 2HPβCD subcutaneously at 49, 56, 63 and 70 days of age, with measurements of organ weights, liver function tests and tissue cholesterol levels performed at 77 days. In Npc1(-/-) mice, treatment with 2HPβCD from 49 days reduced whole-liver cholesterol content at 77 days from 33.0 ± 1.0 to 9.1 ± 0.5 mg/organ. Comparable improvements were seen in other organs, such as the spleen, and in the animal as a whole. There was a transient increase in biliary cholesterol concentration in Npc1(-/-) mice after 2HPβCD. Plasma alanine aminotransferase and aspartate aminotransferase activities in 77-day-old 2HPβCD-treated Npc1(-/-) mice were reduced compared with saline-treated controls. The lifespan of Npc1(-/-) mice given 2HPβCD marginally exceeded that of the saline-treated controls (99 ± 1.1 vs 94 ± 1.4 days, respectively; P < 0.05). Thus, 2HPβCD is effective in mobilizing entrapped cholesterol in late-stage NPC disease leading to improved liver function.

  4. Epitope-tagged dopamine transporter knock-in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons

    PubMed Central

    Rao, Anjali; Richards, Toni L.; Simmons, Diana; Zahniser, Nancy R.; Sorkin, Alexander

    2012-01-01

    The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.—Rao, A., Richards, T. L., Simmons, D., Zahniser, N. R., Sorkin, A. Epitope-tagged dopamine transporter knock-in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons. PMID:22267337

  5. Prenatal exposure of mice to the human liver carcinogen aflatoxin B1 reveals a critical window of susceptibility to genetic change.

    PubMed

    Chawanthayatham, Supawadee; Thiantanawat, Apinya; Egner, Patricia A; Groopman, John D; Wogan, Gerald N; Croy, Robert G; Essigmann, John M

    2015-03-15

    It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B1 (AFB1 ) during prenatal development and the risk of later life genetic disease. AFB1 was given to pregnant C57BL/6J mice, carrying F1 gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-(15) N5 -guanine adduct standards afforded measurement of the AFB1 -N(7) -Gua and AFB1 -FAPY adducts 6-hr post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F1 were analyzed for mutations in the gpt gene at 3 and 10 weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB1 and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB1 . Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated.

  6. Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice.

    PubMed

    Sekerková, Gabriella; Richter, Claus-Peter; Bartles, James R

    2011-03-01

    Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10-0.14 µm, making stereocilia ∼30%-60% thinner than wild type and suggesting that they contained ∼50%-85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the

  7. Temporal dynamics of the developing lung transcriptome in three common inbred strains of laboratory mice reveals multiple stages of postnatal alveolar development

    PubMed Central

    Beauchemin, Kyle J.; Wells, Julie M.; Kho, Alvin T.; Philip, Vivek M.; Kamir, Daniela; Kohane, Isaac S.

    2016-01-01

    To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http

  8. Long-Term Fate Mapping Using Conditional Lentiviral Vectors Reveals a Continuous Contribution of Radial Glia-Like Cells to Adult Hippocampal Neurogenesis in Mice

    PubMed Central

    Aelvoet, Sarah-Ann; Pascual-Brazo, Jesus; Libbrecht, Sarah; Reumers, Veerle; Gijsbers, Rik; Van den Haute, Chris; Baekelandt, Veerle

    2015-01-01

    Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone and the hippocampal dentate gyrus. Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Although tremendous progress has been made in our understanding of adult hippocampal neurogenesis, important questions remain unanswered, regarding the identity and the behavior of neural stem cells in the dentate gyrus. We previously showed that conditional Cre-Flex lentiviral vectors can be used to label neural stem cells in the subventricular zone and to track the migration of their progeny with non-invasive bioluminescence imaging. Here, we applied these Cre-Flex lentiviral vectors to study neurogenesis in the dentate gyrus with bioluminescence imaging and histological techniques. Stereotactic injection of the Cre-Flex vectors into the dentate gyrus of transgenic Nestin-Cre mice resulted in specific labeling of the nestin-positive neural stem cells. The labeled cell population could be detected with bioluminescence imaging until 9 months post injection, but no significant increase in the number of labeled cells over time was observed with this imaging technique. Nevertheless, the specific labeling of the nestin-positive neural stem cells, combined with histological analysis at different time points, allowed detailed analysis of their neurogenic potential. This long-term fate mapping revealed that a stable pool of labeled nestin-positive neural stem cells continuously contributes to the generation of newborn neurons in the mouse brain until 9 months post injection. In conclusion, the Cre-Flex technology is a valuable tool to address remaining questions regarding neural stem cell identity and behavior in the dentate gyrus. PMID:26600383

  9. The Temporal Pattern of Changes in Serum Biomarker Levels Reveals Complex and Dynamically Changing Pathologies after Exposure to a Single Low-Intensity Blast in Mice

    PubMed Central

    Ahmed, Farid; Plantman, Stefan; Cernak, Ibolja; Agoston, Denes V.

    2015-01-01

    Time-dependent changes in blood-based protein biomarkers can help identify the ­pathological processes in blast-induced traumatic brain injury (bTBI), assess injury severity, and monitor disease progression. We obtained blood from control and injured mice (exposed to a single, low-intensity blast) at 2-h, 1-day, 1–week, and 1-month post-injury. We then determined the serum levels of biomarkers related to metabolism (4-HNE, HIF-1α, ceruloplasmin), vascular function (AQP1, AQP4, VEGF, vWF, Flk-1), inflammation (OPN, CINC1, fibrinogen, MIP-1a, OX-44, p38, MMP-8, MCP-1 CCR5, CRP, galectin-1), cell adhesion and the extracellular matrix (integrin α6, TIMP1, TIMP4, Ncad, connexin-43), and axonal (NF-H, Tau), neuronal (NSE, CK-BB) and glial damage (GFAP, S100β, MBP) at various post-injury time points. Our findings indicate that the exposure to a single, low-intensity blast results in metabolic and vascular changes, altered cell adhesion, and axonal and neuronal injury in the mouse model of bTBI. Interestingly, serum levels of several inflammatory and astroglial markers were either unchanged or elevated only during the acute and subacute phases of injury. Conversely, serum levels of the majority of biomarkers related to metabolic and vascular functions, cell adhesion, as well as neuronal and axonal damage remained elevated at the termination of the experiment (1 month), indicating long-term systemic and cerebral alterations due to blast. Our findings show that the exposure to a single, low-intensity blast induces complex pathological processes with distinct temporal profiles. Hence, monitoring serum biomarker levels at various post-injury time points may provide enhanced diagnostics in blast-related neurological and multi-system deficits. PMID:26124743

  10. Type II collagen antibody response is enriched in the synovial fluid of rheumatoid joints and directed to the same major epitopes as in collagen induced arthritis in primates and mice

    PubMed Central

    2014-01-01

    Introduction Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA. Methods Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice. Results Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group. Conclusion CII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation. PMID:25005029

  11. Impaired Oral Tolerance Induction in Diabetes Prone but not in Diabetes Resistant Mice Revealed by Cholera Toxin Subunit B-Peptide Fusion Proteins

    PubMed Central

    Presa, Maximiliano; Ortiz, Angela Zarama; Garabatos, Nahir; Izquierdo, Cristina; Rivas, Elisa I.; Teyton, Luc; Mora, Conchi; Serreze, David; Stratmann, Thomas

    2013-01-01

    The B subunit of cholera toxin (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on antigen-specific CD4 T cells has remained largely unexplored. We investigated by tetramer analysis how oral delivery of CTB fused to 2 CD4 T cell epitopes, the BDC-2.5 T cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4 T cells in NOD mice. CTB-2.5mi activated 2.5mi+ T cells when administered intraperitoneally and generated Ag-specific Foxp3+ Treg and Th2 cells following intragastric delivery. While 2.5mi+ and GAD-specific T cells were tolerized in diabetes resistant NODxB6.Foxp3EGFP F1 and NOR mice, this did not occur in NOD mice. This indicated NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. Contrarily to NODxB6.Foxp3EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi+ T cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients. PMID:23925934

  12. Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors.

    PubMed

    Hansotia, Tanya; Baggio, Laurie L; Delmeire, Dominique; Hinke, Simon A; Yamada, Yuichiro; Tsukiyama, Katsushi; Seino, Yutaka; Holst, Jens J; Schuit, Frans; Drucker, D J

    2004-05-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors.

  13. The erythroid function of transferrin receptor 2 revealed by Tmprss6 inactivation in different models of transferrin receptor 2 knockout mice

    PubMed Central

    Nai, Antonella; Pellegrino, Rosa M.; Rausa, Marco; Pagani, Alessia; Boero, Martina; Silvestri, Laura; Saglio, Giuseppe; Roetto, Antonella; Camaschella, Clara

    2014-01-01

    Transferrin receptor 2 (TFR2) is a transmembrane glycoprotein expressed in the liver and in the erythroid compartment, mutated in a form of hereditary hemochromatosis. Hepatic TFR2, together with HFE, activates the transcription of the iron-regulator hepcidin, while erythroid TFR2 is a member of the erythropoietin receptor complex. The TMPRSS6 gene, encoding the liver-expressed serine protease matriptase-2, is the main inhibitor of hepcidin and inactivation of TMPRSS6 leads to iron deficiency with high hepcidin levels. Here we evaluate the phenotype resulting from the genetic loss of Tmprss6 in Tfr2 total (Tfr2−/−) and liver-specific (Tfr2LCKO) knockout mice. Tmprss6−/−Tfr2−/− and Tmprss6−/−Tfr2LCKO mice have increased hepcidin levels and show iron-deficiency anemia like Tmprss6−/−mice. However, while Tmprss6−/−Tfr2LCKO are phenotypically identical to Tmprss6−/− mice, Tmprss6−/−Tfr2−/− mice have increased red blood cell count and more severe microcytosis than Tmprss6−/− mice. In addition hepcidin expression in Tmprss6−/−Tfr2−/− mice is higher than in the wild-type animals, but lower than in Tmprss6−/− mice, suggesting partial inhibition of the hepcidin activating pathway. Our results prove that hepatic TFR2 acts upstream of TMPRSS6. In addition Tfr2 deletion causes a relative erythrocytosis in iron-deficient mice, which likely attenuates the effect of over-expression of hepcidin in Tmprss6−/− mice. Since liver-specific deletion of Tfr2 in Tmprss6−/− mice does not modify the erythrocyte count, we speculate that loss of Tfr2 in the erythroid compartment accounts for the hematologic phenotype of Tmprss6−/−Tfr2−/− mice. We propose that TFR2 is a limiting factor for erythropoiesis, particularly in conditions of iron restriction. PMID:24658816

  14. Arctigenin but not arctiin acts as the major effective constituent of Arctium lappa L. fruit for attenuating colonic inflammatory response induced by dextran sulfate sodium in mice.

    PubMed

    Wu, Xin; Yang, Yan; Dou, Yannong; Ye, Jun; Bian, Difei; Wei, Zhifeng; Tong, Bei; Kong, Lingyi; Xia, Yufeng; Dai, Yue

    2014-12-01

    The crude powder of the fruit of Arctium lappa L. (ALF) has previously been reported to attenuate experimental colitis in mice. But, its main effective ingredient and underlying mechanisms remain to be identified. In this study, ALF was extracted with ethanol, and then successively fractionated into petroleum ether, ethyl acetate, n-butanol and water fraction. Experimental colitis was induced by dextran sulfate sodium (DSS) in mice. Among the four fractions of ALF, the ethyl acetate fraction showed the most significant inhibition of DSS-induced colitis in mice. The comparative studies of arctigenin and arctiin (the two main ingredients of ethyl acetate fraction) indicated that arctigenin rather than arctiin could reduce the loss of body weight, disease activity index and histological damage in the colon. Arctigenin markedly recovered the loss of intestinal epithelial cells (E-cadherin-positive cells) and decreased the infiltration of neutrophils (MPO-positive cells) and macrophages (CD68-positive cells). Arctigenin could down-regulate the expressions of TNF-α, IL-6, MIP-2, MCP-1, MAdCAM-1, ICAM-1 and VCAM-1 at both protein and mRNA levels in colonic tissues. Also, it markedly decreased the MDA level, but increased SOD activity and the GSH level. Of note, the efficacy of arctigenin was comparable or even superior to that of the positive control mesalazine. Moreover, it significantly suppressed the phosphorylation of MAPKs and the activation of NF-κB, including phosphorylation of IκBα and p65, p65 translocation and DNA binding activity. In conclusion, arctigenin but not arctiin is the main active ingredient of ALF for attenuating colitis via down-regulating the activation of MAPK and NF-κB pathways.

  15. Identity of major sulfur-cycle prokaryotes in freshwater lake ecosystems revealed by a comprehensive phylogenetic study of the dissimilatory adenylylsulfate reductase

    PubMed Central

    Watanabe, Tomohiro; Kojima, Hisaya; Fukui, Manabu

    2016-01-01

    Adenylylsulfate reductase is a heterodimeric complex of two subunits, AprB and AprA, and is a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. Common use of aprA as a functional marker gene has revealed the diversity of sulfur-cycle prokaryotes in diverse environments. In this study, we established a comprehensive sequence set of apr genes and employed it to reanalyze apr phylogeny, evaluate the coverage of a widely used primer set (AprA-1-FW/AprA-5-RV), and categorize environmental aprA sequences. Phylogenetic tree construction revealed new members of Apr lineage II and several previously unrecognized lateral gene transfer events. Using the established phylogenetic tree, we classified all previously reported aprA sequences amplified from freshwater lakes with the primer pair AprA-1-FW/AprA-5-RV in addition to the aprA sequences newly retrieved from freshwater lakes; the obtained results were complemented by 16S rRNA clone library analysis. Apr-based classifications of some of operational taxonomic units were supported by 16S rRNA-based analysis. This study updates our knowledge on the phylogeny of aprBA and shows the identities of several sulfur-cycle bacteria, which could not be classified to a known taxa until now. The established apr sequence set is publicly available and can be applied to assign environmental sequences to known lineages. PMID:27824124

  16. Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans.

    PubMed

    Dumesic, Phillip A; Rosenblad, Magnus A; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J; Yates, John R; Madhani, Hiten D

    2015-10-15

    Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features-SRP RNA helix 8 and Srp54-in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development.

  17. Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

    PubMed Central

    Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

    2015-01-01

    Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

  18. Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

    PubMed Central

    2012-01-01

    Background The critical role of Major Histocompatibility Complex (Mhc) genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major) from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help understanding how parasite

  19. Sodium cromoglycate inhibits absorption of the major soybean allergen, Gly m Bd 30K, in mice and human intestinal Caco-2 cells.

    PubMed

    Weangsripanaval, Thanakorn; Murota, Kaeko; Murakami, Yoko; Kominami, Masaru; Kusudo, Tatsuya; Moriyama, Tatsuya; Ogawa, Tadashi; Kawada, Teruo

    2006-11-01

    Our previous data showed that Gly m Bd 30K was absorbed from the gastrointestinal tract and circulated in blood in mice. This study was conducted to determine the mechanism and identify the inhibitor of such absorption. Using sandwich ELISA and immunoblotting, we found that intact Gly m Bd 30K was absorbed from apical to basolateral solutions and intracellularly accumulated by Caco-2 cells in a dose- and time-dependent manner. The absorption and intracellular accumulation of Gly m Bd 30K were significantly suppressed when Caco-2 cells were treated with sodium cromoglycate (SCG) (0-50 mmol/L) in a dose-dependent manner. In 24-d-old mice orally treated with SCG (10-1000 mg/kg body weight), plasma Gly m Bd 30K concentration decreased significantly 30-120 min after Gly m Bd 30K (2000 mg/kg body weight) administration. Moreover, inhibitors that suppress the clathrin-dependent endocytosis dansylcadaverine, the caveolae-dependent endocytosis nystatin and clathrin, and the caveolae-dependent endocytosis methyl-beta-cyclodextrin had inhibitory effects on the absorption and intracellular accumulation of Gly m Bd 30K by Caco-2 cells. These data indicate that Gly m Bd 30K is absorbed and intracellularly accumulated in Caco-2 cells via clathrin- or caveolae-dependent endocytosis. We propose that the absorption and intracellular accumulation of Gly m Bd 30K are inhibited by SCG via clathrin- or caveolae-dependent endocytosis.

  20. Systematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast.

    PubMed

    Narayanaswamy, Rammohan; Moradi, Emily K; Niu, Wei; Hart, G Traver; Davis, Matthew; McGary, Kriston L; Ellington, Andrew D; Marcotte, Edward M

    2009-01-01

    Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.

  1. Irreversible blockade of monoamine oxidases reveals the critical role of 5-HT transmission in locomotor response induced by nicotine in mice.

    PubMed

    Villégier, Anne-Sophie; Salomon, Lucas; Blanc, Gérard; Godeheu, Gérard; Glowinski, Jacques; Tassin, Jean-Pol

    2006-09-01

    Although nicotine is generally considered as the main compound responsible for addictive properties of tobacco, some experimental data indicate that nicotine does not exhibit all the characteristics of other substances of misuse such as psychostimulants and opiates. For example, nicotine generally fails to induce locomotor response in mice and self-administration of nicotine is difficult to obtain in rats. We have shown recently that a pretreatment with mixed irreversible monoamine oxidase inhibitors (MAOIs), such as tranylcypromine, triggers a locomotor response to nicotine in mice and induces a robust self-administration of nicotine in rats. We show here that when mice were pretreated with enhancers of extracellular levels of noradrenaline, dopamine or serotonin (D-amphetamine, GBR12783 or para-chloro-amphetamine, respectively) and injected with nicotine (1 mg/kg), only those animals pretreated with para-chloro-amphetamine exhibited a specific locomotor response to nicotine. These data indicate a critical role of serotonin in nicotine-induced locomotor activity in mice. This was further confirmed in microdialysis experiments showing that nicotine induces an increase in extracellular serotonin levels in the ventral striatum in mice pretreated with tranylcypromine. This effect of nicotine on extracellular serotonin levels was absent in mice lacking the beta2-subunit of the nicotinic acetylcholine receptor. Our data suggest that mixed irreversible MAOIs contained in tobacco facilitate the effects of nicotine on serotonin release, thus allowing the locomotor and rewarding effects of nicotine.

  2. Dissection of Genomewide-Scan Data in Extended Families Reveals a Major Locus and Oligogenic Susceptibility for Age-Related Macular Degeneration

    PubMed Central

    Iyengar, Sudha K.; Song, Danhong; Klein, Barbara E. K.; Klein, Ronald; Schick, James H.; Humphrey, Jennifer; Millard, Christopher; Liptak, Rachel; Russo, Karlie; Jun, Gyungah; Lee, Kristine E.; Fijal, Bonnie; Elston, Robert C.

    2004-01-01

    To examine the genetic basis of age-related macular degeneration (ARMD), a degenerative disease of the retinal pigment epithelium and neurosensory retina, we conducted a genomewide scan in 34 extended families (297 individuals, 349 sib pairs) ascertained through index cases with neovascular disease or geographic atrophy. Family and medical history was obtained from index cases and family members. Fundus photographs were taken of all participating family members, and these were graded for severity by use of a quantitative scale. Model-free linkage analysis was performed, and tests of heterogeneity and epistasis were conducted. We have evidence of a major locus on chromosome 15q (GATA50C03 multipoint P=1.98×10-7; empirical P⩽1.0×10-5; single-point P=3.6×10-7). This locus was present as a weak linkage signal in our previous genome scan for ARMD, in the Beaver Dam Eye Study sample (D15S659, multipoint P=.047), but is otherwise novel. In this genome scan, we observed a total of 13 regions on 11 chromosomes (1q31, 2p21, 4p16, 5q34, 9p24, 9q31, 10q26, 12q13, 12q23, 15q21, 16p12, 18p11, and 20q13), with a nominal multipoint significance level of P⩽.01 or LOD ⩾1.18. Family-by-family analysis of the data, performed using model-free linkage methods, suggests that there is evidence of heterogeneity in these families. For example, a single family (family 460) individually shows linkage evidence at 8 loci, at the level of P<.0001. We conducted tests for heterogeneity, which suggest that ARMD susceptibility loci on chromosomes 9p24, 10q26, and 15q21 are not present in all families. We tested for mutations in linked families and examined SNPs in two candidate genes, hemicentin-1 and EFEMP1, in subsamples (145 and 189 sib pairs, respectively) of the data. Mutations were not observed in any of the 11 exons of EFEMP1 nor in exon 104 of hemicentin-1. The SNP analysis for hemicentin-1 on 1q31 suggests that variants within or in very close proximity to this gene cause ARMD

  3. The Shigella dysenteriae serotype 1 proteome, profiled in the host intestinal environment, reveals major metabolic modifications and increased expression of invasive proteins

    PubMed Central

    Pieper, Rembert; Zhang, Quanshun; Parmar, Prashanth P.; Huang, Shih-Ting; Clark, David J.; Alami, Hamid; Donohue-Rolfe, Arthur; Fleischmann, Robert D.; Peterson, Scott N.; Tzipori, Saul

    2010-01-01

    Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system (T3SS) effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the T3SS effectors OspC2 and IpaB, were detected as antigens in western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets. PMID:19813213

  4. Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

    PubMed Central

    Gramaje, David; León, Maela; Santana, Marcela; Crous, Pedro W.; Armengol, Josep

    2014-01-01

    Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions. PMID:25310345

  5. Multilocus ISSR markers reveal two major genetic groups in Spanish and South African populations of the grapevine fungal pathogen Cadophora luteo-olivacea.

    PubMed

    Gramaje, David; León, Maela; Santana, Marcela; Crous, Pedro W; Armengol, Josep

    2014-01-01

    Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.

  6. Task and Resting-State fMRI Reveal Altered Salience Responses to Positive Stimuli in Patients with Major Depressive Disorder

    PubMed Central

    Yang, Yang; Zhong, Ning; Imamura, Kazuyuki; Lu, Shengfu; Li, Mi; Zhou, Haiyan; Li, Huaizhou; Yang, Xiaojing; Wan, Zhijiang; Wang, Gang; Hu, Bin; Li, Kuncheng

    2016-01-01

    Altered brain function in patients with major depressive disorder (MDD) has been repeatedly demonstrated by task-based and resting-state studies, respectively. However, less is known concerning whether overlapped abnormalities in functional activities across modalities exist in MDD patients. To find out the answer, we implemented an fMRI experiment and collected both task and resting-state data from 19 MDD patients and 19 matched, healthy, controls. A distraction paradigm involving emotionally valenced pictures was applied to induce affective responses in subjects. As a result, concurrent deficits were found in arousing activation during a positive task in both the reward circuit and salience network (SN) that is composed of the dorsal part of anterior cingulate cortex (dACC) and bilateral anterior insulae (AI) in only the MDD group. Subsequent amplitude of low frequency fluctuations (ALFF) and functional connectivity analyses based on resting-state data exhibited consistent alterations in the bilateral AI of MDD patients, and indicated patients’ difficulties in regulating the balance between central executive network (CEN) and default mode network (DMN) due to altered connectivity among the CEN, DMN, and SN. Our findings provide new evidence demonstrating impaired salience processing and resulting alterations in responses to positive stimuli in MDD patients. Furthermore, brain abnormalities synchronized across functional states in MDD patients can be evidenced by a combination of task and resting-state fMRI analyses. PMID:27192082

  7. Fold-to-fault progression of a major thrust zone revealed in horses of the North Mountain fault zone, Virginia and West Virginia, USA

    USGS Publications Warehouse

    Orndorff, Randall C.

    2012-01-01

    The method of emplacement and sequential deformation of major thrust zones may be deciphered by detailed geologic mapping of these important structures. Thrust fault zones may have added complexity when horse blocks are contained within them. However, these horses can be an important indicator of the fault development holding information on fault-propagation folding or fold-to-fault progression. The North Mountain fault zone of the Central Appalachians, USA, was studied in order to better understand the relationships of horse blocks to hanging wall and footwall structures. The North Mountain fault zone in northwestern Virginia and eastern panhandle of West Virginia is the Late Mississippian to Permian Alleghanian structure that developed after regional-scale folding. Evidence for this deformation sequence is a consistent progression of right-side up to overturned strata in horses within the fault zone. Rocks on the southeast side (hinterland) of the zone are almost exclusively right-side up, whereas rocks on the northwest side (foreland) of the zone are almost exclusively overturned. This suggests that the fault zone developed along the overturned southeast limb of a syncline to the northwest and the adjacent upright limb of a faulted anticline to the southeast.

  8. Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes-prone but not in diabetes-resistant mice.

    PubMed

    Presa, Maximiliano; Ortiz, Angela Zarama; Garabatos, Nahir; Izquierdo, Cristina; Rivas, Elisa I; Teyton, Luc; Mora, Conchi; Serreze, David; Stratmann, Thomas

    2013-11-01

    The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag-specific CD4(+) T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4(+) T-cell epitopes, the BDC-2.5 T-cell 2.5 mi mimotope and glutamic acid decarboxylase (GAD) 286-300, affected diabetogenic CD4(+) T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB-2.5 mi activated 2.5 mi(+) T cells and following intragastric delivery generated Ag-specific Foxp3(+) Treg and Th2 cells. While 2.5 mi(+) and GAD-specific T cells were tolerized in diabetes-resistant NODxB6.Foxp3(EGFP) F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. In contrast to NODxB6.Foxp3(EGFP) F1 mice, oral treatment in NOD mice lead to strong 2.5 mi(+) T-cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5 mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.

  9. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome.

    PubMed

    Jaafari, Mahmoud R; Badiee, Ali; Khamesipour, Ali; Samiei, Afshin; Soroush, Dina; Kheiri, Masoumeh Tavassoti; Barkhordari, Farzaneh; McMaster, W Robert; Mahboudi, Fereidoun

    2007-08-10

    CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.

  10. Collagen induced arthritis (CIA) in mice features regulatory transcriptional network connecting major histocompatibility complex (MHC H2) with autoantigen genes in the thymus.

    PubMed

    Donate, Paula B; Fornari, Thaís A; Junta, Cristina M; Magalhães, Danielle A; Macedo, Cláudia; Cunha, Thiago M; Nguyen, Catherine; Cunha, Fernando Q; Passos, Geraldo A

    2011-05-01

    Considering that imbalance of central tolerance in the thymus contributes to aggressive autoimmunity, we compared the expression of peripheral tissue autoantigens (PTA) genes, which are involved in self-representation in the thymic stroma, of two mouse strains; DBA-1/J (MHC-H2(q)) susceptible and DBA-2/J (MHC-H2(d)) resistant to collagen induced arthritis (CIA). We evaluate whether these strains differ in their thymic gene expression, allowing identification of genes that might play a role in susceptibility/resistance to CIA. Microarray profiling showed that 1093 PTA genes were differentially modulated between collagen immunized DBA-1/J and DBA-2/J mice. These genes were assigned to 17 different tissues/organs, including joints/bone, characterizing the promiscuous gene expression (PGE), which is implicated in self-representation. Hierarchical clustering of microarray data and quantitative RT-PCR analysis showed that Aire (autoimmune regulator), an important regulator of the PGE process, Aire-dependent (insulin), Aire-independent (Col2A1 and Gad67), and other 22 joint/bone autoantigen genes were down-regulated in DBA-1/J compared with DBA-2/J in the thymus. Considering the importance of MHC-H2 in peptide-self presentation and autoimmunity susceptibility, we reconstructed transcriptional networks of both strains based on actual microarray data. The networks clearly demonstrated different MHC-H2 transcriptional interactions with PTAs genes. DBA-1/J strain featured MHC-H2 as a node influencing downstream genes. Differently, in DBA-2/J strain network MHC-H2 was exclusively self-regulated and does not control other genes. These findings provide evidence that CIA susceptibility in mice may be a reflex of a cascade-like transcriptional control connecting different genes to MHC-H2 in the thymus.

  11. CD25+CD127+Foxp3- Cells Represent a Major Subpopulation of CD8+ T Cells in the Eye Chambers of Normal Mice

    PubMed Central

    Ziółkowska, Natalia; Ziółkowski, Hubert; Małaczewska, Joanna

    2017-01-01

    The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege. PMID:28081241

  12. The evolution of the spindlin gene in birds: sequence analysis of an intron of the spindlin W and Z gene reveals four major divisions of the Psittaciformes.

    PubMed

    de Kloet, Rolf S; de Kloet, Siwo R

    2005-09-01

    The Psittaciformes (parrots, parakeets) are among the most widely held captive birds. Yet, their evolution and their phylogenetic relationships have been relatively little studied. This paper describes the phylogenetic relationships between a number of Psittaciformes as derived from the sequences of the third intron of the Z-chromosomal and W-chromosomal spindlin genes. The Z-chromosomal sequences of the kakapo (Strigops habroptilus), the kea (Nestor notabilis), and the kaka (Nestor meridionalis) from New Zealand form a cluster which is the sister group to all other Psittaciformes. The results show further that the Z-chromosomal sequences of the other species can be divided into two groups based on the occurrence of a sequence element ACCCT. The group with the insert (A) is mainly from species with an Australasian geographical distribution and includes such species as the Lories (Lorius, etc.), the budgerigar (Melospittacus undulatus), and the rosellas (Platycercus). It also includes the African lovebirds (Agapornidae), which are the only representative of group A outside Australasia. Group B, without the insert, includes the neotropical parrots and parakeets such as the amazons (Amazona, etc.), the macaws (Ara, etc.), and the conures (Aratinga, etc.), the Australian Cacatuini and the African species such as the African grey parrot (Psittacus erithacus) as well as Coracopsis vasa from Madagascar and Psittrichas fulgidus from New Guinea. The W-chromosomal sequence data show that another division of the Psittacidae is found in the replacement of a pyrimidine-rich segment occurring in many non-psittacines as well as the kakapo (S. habroptilus), the kea (N. notabilis), the kaka (N. meridionalis), and the Cacatuini by a microsatellite consisting of a variable number of TATTA monomers in the other Psittaciformes. The results support a Gondwanan origin of the Psittaciformes and the suggestion that paleogeographic events were a major force in psittacine divergence.

  13. A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome.

    PubMed

    Cuykendall, Tawny N; Satyaki, P; Ji, Shuqing; Clay, Derek M; Edelman, Nathaniel B; Kimchy, Alexandra; Li, Ling-Hei; Nuzzo, Erin A; Parekh, Neil; Park, Suna; Barbash, Daniel A

    2014-10-27

    Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality.

  14. Human-Mouse Chimeras With Normal Expression and Function Reveal That Major Domain Swapping is Tolerated by P-glycoprotein (ABCB1)

    PubMed Central

    Pluchino, Kristen M.; Hall, Matthew D.; Moen, Janna K.; Chufan, Eduardo E.; Fetsch, Patricia A.; Shukla, Suneet; Gill, Deborah R.; Hyde, Stephen C.; Xia, Di; Ambudkar, Suresh V.; Gottesman, Michael M.

    2017-01-01

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-crosslinking with [125I]-Iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug-binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes. PMID:26820614

  15. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  16. Comparative Developmental Transcriptomics Reveals Rewiring of a Highly Conserved Gene Regulatory Network during a Major Life History Switch in the Sea Urchin Genus Heliocidaris

    PubMed Central

    Israel, Jennifer W.; Martik, Megan L.; Byrne, Maria; Raff, Elizabeth C.; Raff, Rudolf A.; McClay, David R.; Wray, Gregory A.

    2016-01-01

    The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results

  17. Long-Term MR Cell Tracking of Neural Stem Cells Grafted in Immunocompetent Versus Immunodeficient Mice Reveals Distinct Differences in Contrast Between Live and Dead Cells

    PubMed Central

    Berman, Stacey Cromer; Galpoththawela, Chulani; Gilad, Assaf A.; Bulte, Jeff W. M.; Walczak, Piotr

    2011-01-01

    Neural stem cell (NSC)-based therapy is actively being pursued in preclinical and clinical disease models. Magnetic resonance imaging (MRI) cell tracking promises to optimize current cell transplantation paradigms, however, it is limited by dilution of contrast agent during cellular proliferation, transfer of label from dying cells to surrounding endogenous host cells, and/or biodegradation of the label. Here, we evaluated the applicability of magnetic resonance imaging for long-term tracking of transplanted neural stem cells labeled with superparamagnetic iron oxide and transfected with the bioluminescence reporter gene luciferase. Mouse neural stem cells were transplanted into immunodeficient, graft-accepting Rag2 mice or immunocompetent, graft-rejecting Balb/c mice. Hypointense voxel signals and bioluminescence were monitored over a period of 93 days. Unexpectedly, in mice that rejected the cells, the hypointense MR signal persisted throughout the entire time-course, whereas in the nonrejecting mice, the contrast cleared at a faster rate. In immunocompetent, graft-rejecting Balb/c mice, infiltrating leukocytes, and microglia were found surrounding dead cells and internalizing superparamagnetic iron oxide clusters. The present results indicate that live cell proliferation and associated label dilution may dominate contrast clearance as compared with cell death and subsequent transfer and retention of superparamagnetic iron oxide within phagocytes and brain interstitium. Thus, interpretation of signal changes during long-term MR cell tracking is complex and requires caution. PMID:20928883

  18. Host transcriptomic responses to pneumonic plague reveal that Yersinia pestis inhibits both the initial adaptive and innate immune responses in mice.

    PubMed

    Yang, Huiying; Wang, Tong; Tian, Guang; Zhang, Qingwen; Wu, Xiaohong; Xin, Youqian; Yan, Yanfeng; Tan, Yafang; Cao, Shiyang; Liu, Wanbing; Cui, Yujun; Yang, Ruifu; Du, Zongmin

    2017-01-01

    Pneumonic plague is the most deadly form of infection caused by Yersinia pestis and can progress extremely fast. However, our understanding on the host transcriptomic response to pneumonic plague is insufficient. Here, we used RNA-sequencing technology to analyze transcriptomic responses in mice infected with fully virulent strain 201 or EV76, a live attenuated vaccine strain lacking the pigmentation locus. Approximately 600 differentially expressed genes (DEGs) were detected in lungs from both 201- and EV76-infected mice at 12h post-infection (hpi). DEGs in lungs of 201-infected mice exceeded 2000 at 48hpi, accompanied by sustained large numbers of DEGs in the liver and spleen; however, limited numbers of DEGs were detected in those organs of EV-infected mice. Remarkably, DEGs in lungs were significantly enriched in critical immune responses pathways in EV76-infected but not 201-infected mice, including antigen processing and presentation, T cell receptor signaling among others. Pathological and bacterial load analyses confirmed the rapid systemic dissemination of 201-infection and the confined EV76-infection in lungs. Our results suggest that fully virulent Y. pestis inhibits both the innate and adaptive immune responses that are substantially stimulated in a self-limited infection, which update our holistic views on the transcriptomic response to pneumonic plague.

  19. Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

    SciTech Connect

    Petrus, Amanda K.; Swithers, Kristen S.; Ranjit, Chaman R.; Wu, Si; Brewer, Heather M.; Gogarten, J Peter; Pasa-Tolic, Ljiljana; Noll, Kenneth M.

    2012-06-29

    The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompa) and the porin OmpB (or Ompb). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant b-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had b-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.

  20. Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

    PubMed Central

    Petrus, Amanda K.; Swithers, Kristen S.; Ranjit, Chaman; Wu, Si; Brewer, Heather M.; Gogarten, J. Peter; Pasa-Tolic, Ljiljana; Noll, Kenneth M.

    2012-01-01

    The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath. PMID:22768259

  1. Genes for the major structural components of Thermotogales species' togas revealed by proteomic and evolutionary analyses of OmpA and OmpB homologs.

    PubMed

    Petrus, Amanda K; Swithers, Kristen S; Ranjit, Chaman; Wu, Si; Brewer, Heather M; Gogarten, J Peter; Pasa-Tolic, Ljiljana; Noll, Kenneth M

    2012-01-01

    The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.

  2. Complete amino acid sequence of heavy chain variable regions derived from two monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain

    PubMed Central

    1984-01-01

    The primary structure of A/J anti-p-azophenylarsonate (anti-Ars) antibodies expressing the major A-strain cross-reactive idiotype (CRIA) has provided important insights into issues of antibody diversity and the molecular basis of idiotypy in this important model system. Until recently, this idiotype was thought to be rarely, if ever, expressed in BALB/c mice. Indeed, it has been reported that BALB/c mice lack the heavy chain variable segment (VH) gene that is utilized by the entire family of anti-Ars antibodies expressing the A/J CRI. Recently, however, it has been possible to elicit CRIA+, Ars binding antibodies in the BALB/c strain by immunizing first with anti-CRI and then with antigen. Such BALB/c, CRIA+ anti-Ars antibodies can be induced occasionally with antigen alone. VH region amino acid sequences are described for two CRIA+ hybridoma products derived from BALB/c mice. While remarkably similar to each other, their VH segments (1-98) differ from the VH segments of A/J CRIA+, anti-Ars antibodies in over 40 positions. Rather than the usual JH2 gene segment used by most A/J CRIA+ anti-Ars antibodies, one BALB/c CRIA+ hybridoma utilizes a JH1 gene segment, while the other uses a JH4. However, the D segments of both of the BALB/c antibodies are remarkably homologous to the D segments of several A/J CRIA+ antibodies sequenced previously, as are the amino terminal amino acid sequences of their light chains. These data imply that BALB/c mice express the A/J CRIA by producing antibodies with very similar, if not identical, light chain and heavy chain D segments, but in the context of different VH and JH gene segments than their A/J counterparts. The results document that molecules that share serologic specificities can have vastly different primary structures. PMID:6207261

  3. Major- and trace-elements in cratonic mantle eclogites and pyroxenites reveal heterogeneous sources and metamorphic processing of low-pressure protoliths

    NASA Astrophysics Data System (ADS)

    Aulbach, Sonja; Jacob, Dorrit E.

    2016-10-01

    There is a growing body of evidence for the origin of cratonic mantle eclogite xenoliths by low-pressure formation in now-recycled ocean floors. Because they have protoliths ultimately derived from the convecting mantle, their study can potentially yield unprecedented insights into as yet little-understood palaeo-geodynamic regimes, once primary (fractional crystallisation, accumulation, mixing) and secondary processes (kimberlite infiltration, metasomatism) affecting their compositions are understood. This is achieved using diagnostic concentrations or ratios of the analytically and geologically most robust elements (major and minor elements, transition metals, REE), and aided by comparison to natural and modelled analogues. Here, mineral compositions taken from the literature were used to reconstruct bulk rocks and assign the samples to eclogites (further divided into high-Mg, low-Mg and high-Ca types), pyroxenites and their gabbroic (Eu* > 1.05) counterparts. Various protolith types - formed predominantly by < 1 GPa crystallisation from broadly picritic magmas leaving garnet-poor mantle sources - are identified: (1) Many high-Mg eclogites lie on modelled crystallisation trends between 0.5 and < 1 GPa. Some have elevated FeO contents with lower SiO2 and CaO possibly requiring Fe-rich pyroxenite heterogeneities in their mantle source. (2) Many high-Ca eclogites may be the differentiated (higher Na2O, TiO2 and FeO at lower MgO) equivalents of high-Mg eclogites, following modelled crystallisation trends at somewhat lower pressure (0.05 to 0.5 GPa). Other high-Ca eclogites with low FeO were produced during interaction with fluids and melts in mélange-type settings. (3) Low-Mg eclogites, with intermediate MgO content, are too FeO-rich to be intermediary crystallisation products of the same parental melt and are ascribed to melting out of Fe-rich lithologies possibly related to recycling of eclogite and/or contamination with ferromanganese sediments. (4) The positive

  4. Revealing the significance and polyphase tectonothermal evolution of a major metamorphic unit in an orogen: the central Sanandaj-Sirjan zone, Zagros Mts., Iran

    NASA Astrophysics Data System (ADS)

    Shakerardakani, Farzaneh; Neubauer, Franz; Genser, Johann; Liu, Xiaoming; Dong, Yunpeng; Monfaredi, Behzad; Benroider, Manfred; Finger, Fritz; Waitzinger, Michael

    2016-04-01

    The Dorud-Azna region in the central Sanandaj-Sirjan metamorphic belt plays a key role in promoting the tectonic evolution of Zagros orogen, within the frame of the Arabia-Eurasia collision zone. From footwall to hangingwall, structural data combined with the U-Pb zircon and extensive 40Ar-39Ar mineral dating survey demonstrate three metamorphosed tectonic units, which include: (1) The Triassic June complex is metamorphosed within greenschist facies conditions, overlain by (2) the amphibolite-grade metamorphic Galeh-Doz orthogneiss, which is intruded by mafic dykes, and (3) the Amphibolite-Metagabbro unit. To the east, these units were intruded by the Jurassic Darijune gabbro. We present U-Pb detrital zircon ages of a garnet-micaschist from the Amphibolite-Metagabbro unit, which yield six distinctive age groups, including a previously unrecognized Late Grenvillian age population at ~0.93 to 0.99 Ga. We speculate that this unique Late Grenvillian group coupled with biogeographic evidence suggests either relationship with the South China craton or to the "Gondwana superfan". The laser ablation ICP-MS U-Pb zircon ages of 608 ± 18 Ma and 588 ± 41 Ma of the granitic Galeh-Doz orthogneiss reveals a Panafrican basement same as known from the Yazd block of Central Iran. Geochemistry and Sr-Nd isotopes of alkaline and subalkaline mafic dykes within the Galeh-Doz orthogneiss show OIB-type to MORB-type and indicate involvement of both depleted and enriched sources for its genesis. The new 40Ar-39Ar amphibole age of ca. 322.2 ± 3.9 Ma from the alkaline mafic dyke implies Carboniferous cooling age after intrusion. The metagabbros (including the Dare-Hedavand metagabbro with a 206Pb/238U age of 314.6 ± 3.7 Ma) and amphibolites with E-MORB geochemical signature of the Amphibolite-Metagabbro unit represent an Upper Paleozoic rift. The geochemical composition of the Triassic greenschist facies metamorphosed June complex, implying formation in a same, but younger tectonic

  5. Age-dependent regulation of depression-like behaviors through modulation of adrenergic receptor α₁A subtype expression revealed by the analysis of interleukin-1 receptor antagonist knockout mice.

    PubMed

    Wakabayashi, C; Kiyama, Y; Kunugi, H; Manabe, T; Iwakura, Y

    2011-09-29

    Interleukin-1 (IL-1) plays a crucial role in stress responses and its mRNA is induced in the brain by stress load; however, the precise role of IL-1 in higher brain functions and their abnormalities is largely unknown. Here, we report that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lack IL-1Ra molecules that antagonize the IL-1 receptor, displayed anti-depression-like phenotypes in the tail suspension test (TST) and forced-swim test (FST) only at a young stage (8 weeks), whereas the phenotypes disappeared at later stages (20 and 32 weeks). These anti-depression-like phenotypes were reversed by administration of adrenergic receptor (AR) antagonists against the ARα(1), ARα(2), and ARβ subtypes. Although the contents of 5-HT, norepinephrine (NE), and dopamine (DA), which are known to be associated with major symptoms of psychiatric disorders, were not significantly different in the hippocampus or cerebral cortex between IL-1Ra KO and their wild-type (WT) littermate mice, the mRNA expression level of the ARα(1A) subtype was significantly changed in the cerebral cortex. Interestingly, the change in expression of the ARα(1A) subtype was correlated with an age-dependent alteration in the TST and FST in IL-1Ra KO mice. Furthermore, mild immobilization stress loaded on C57BL/6J male mice caused similar anti-depression-like phenotypes in the TST and FST to those observed in mutant mice. These results suggest that sustained activation of IL-1 signaling induced by gene manipulation in mutant mice affects the expression of the ARα(1A) subtype and that modification of adrenergic signaling by the IL-1 system may ultimately cause significant psychiatric abnormalities such as depression, and this mutant mouse could be regarded as a model animal of depression that specifically appears in children and adolescents.

  6. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves.

    PubMed

    Colom, Bartomeu; Poitelon, Yannick; Huang, Wenlong; Woodfin, Abigail; Averill, Sharon; Del Carro, Ubaldo; Zambroni, Desirée; Brain, Susan D; Perretti, Mauro; Ahluwalia, Amrita; Priestley, John V; Chavakis, Triantafyllos; Imhof, Beat A; Feltri, M Laura; Nourshargh, Sussan

    2012-03-01

    Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.

  7. Neutrophil Emigration in the Skin, Lungs, and Peritoneum: Different Requirements for CD11/CD18 Revealed by CD18-deficient Mice

    PubMed Central

    Mizgerd, Joseph P.; Kubo, Hiroshi; Kutkoski, Gregory J.; Bhagwan, Sabrina D.; Scharffetter-Kochanek, Karin; Beaudet, Arthur L.; Doerschuk, Claire M.

    1997-01-01

    To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18−/− mutants). Peripheral blood of CD18−/− mutants contained 11-fold more neutrophils than did blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less in CD18−/− mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil emigration in CD18−/− mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18−/− mutants, but rather was greater than the WT values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate (90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18−/− mutant mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia or peritonitis. PMID:9334375

  8. Genetic manipulation of the ghrelin signaling system in male mice reveals bone compartment specificity of acylated and unacylated ghrelin in the regulation of bone remodeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ghrelin receptor-deficient (Ghsr-/-) mice that lack acylated ghrelin (AG) signaling retain a metabolic response to unacylated ghrelin (UAG). Recently, we showed that Ghsr-deficiency affects bone metabolism. The aim of this study was to further establish the impact of AG and UAG on bone metabolism. W...

  9. Genetic control of a central pattern generator: rhythmic oromotor movement in mice is controlled by a major locus near Atp1a2.

    PubMed

    Boughter, John D; Mulligan, Megan K; St John, Steven J; Tokita, Kenichi; Lu, Lu; Heck, Detlef H; Williams, Robert W

    2012-01-01

    Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h(2)≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM = 116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F(1) hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na(+)/K(+)-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo.

  10. Genetic Control of a Central Pattern Generator: Rhythmic Oromotor Movement in Mice Is Controlled by a Major Locus near Atp1a2

    PubMed Central

    Boughter, John D.; Mulligan, Megan K.; St. John, Steven J.; Tokita, Kenichi; Lu, Lu; Heck, Detlef H.; Williams, Robert W.

    2012-01-01

    Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h2≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM = 116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F1 hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na+/K+-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo. PMID:22675444

  11. Readaptation of a low-virulence influenza H9 escape mutant in mice: the role of changes in hemagglutinin as revealed by site-specific mutagenesis.

    PubMed

    Ilyushina, N A; Rudneva, I A; Khalenkov, A M; Timofeeva, T A; Krylov, P S; Webster, R G; Kaverin, N V

    2010-01-01

    In our earlier studies, we showed that an escape mutant of mouse-adapted H9N2 influenza virus carrying a T198N amino acid change in heamagglutinin (HA) has a lowered virulence for mice. The readaptation of this mutant to mice was associated with N198S or N198D reverse mutations. In this study, single-gene reassortants having HA gene of the wild-type virus, its low-virulence escape mutant, or a readapted variant were generated by site-specific mutagenesis and assayed for virulence. The results showed that antibody-selected mutations in the HA of H9 influenza virus can decrease mortality and virus accumulation in mouse lungs, though not in nasal turbinates, and the effect may be compensated by reverse mutations in the course of passaging.

  12. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development.

    PubMed

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-12-05

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.

  13. Differential Proteomic Analysis of the Pancreas of Diabetic db/db Mice Reveals the Proteins Involved in the Development of Complications of Diabetes Mellitus

    PubMed Central

    Pérez-Vázquez, Victoriano; Guzmán-Flores, Juan M.; Mares-Álvarez, Daniela; Hernández-Ortiz, Magdalena; Macías-Cervantes, Maciste H.; Ramírez-Emiliano, Joel; Encarnación-Guevara, Sergio

    2014-01-01

    Type 2 diabetes mellitus is characterized by hyperglycemia and insulin-resistance. Diabetes results from pancreatic inability to secrete the insulin needed to overcome this resistance. We analyzed the protein profile from the pancreas of ten-week old diabetic db/db and wild type mice through proteomics. Pancreatic proteins were separated in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significant changes in db/db mice respect to wild type mice were observed in 27 proteins. Twenty five proteins were identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and their interactions were analyzed using search tool for the retrieval of interacting genes/proteins (STRING) and database for annotation, visualization and integrated discovery (DAVID). Some of these proteins were Pancreatic α-amylase, Cytochrome b5, Lithostathine-1, Lithostathine-2, Chymotrypsinogen B, Peroxiredoxin-4, Aspartyl aminopeptidase, Endoplasmin, and others, which are involved in the metabolism of carbohydrates and proteins, as well as in oxidative stress, and inflammation. Remarkably, these are mostly endoplasmic reticulum proteins related to peptidase activity, i.e., they are involved in proteolysis, glucose catabolism and in the tumor necrosis factor-mediated signaling pathway. These results suggest mechanisms for insulin resistance, and the chronic inflammatory state observed in diabetes. PMID:24886809

  14. Altered Macrophage and Dendritic Cell Response in Mif−/− Mice Reveals a Role of Mif for Inflammatory-Th1 Response in Type 1 Diabetes

    PubMed Central

    Vazquez-Mendoza, Alicia

    2016-01-01

    Macrophage migration inhibitory factor (Mif) is highly expressed in type 1 diabetes mellitus (T1DM). However, there is limited information about how Mif influences the activation of macrophages (Mφ) and dendritic cells (DC) in T1DM. To address this issue, we induced T1DM by administering multiple low doses of streptozotocin (STZ) to Mif−/− or wild-type (Wt) BALB/c mice. We found that Mif−/− mice treated with STZ (Mif−/−STZ) developed lower levels of hyperglycemia, inflammatory cytokines, and specific pancreatic islet antigen- (PIAg-) IgG and displayed reduced cellular infiltration into the pancreatic islets compared to Wt mice treated with STZ (WtSTZ). Moreover, Mφ and DC from Mif−/−STZ displayed lower expression of MHC-II, costimulatory molecules CD80, CD86, and CD40, Toll-like receptor- (TLR-) 2, and TLR-4 than WtSTZ. These changes were associated with a reduced capacity of Mφ and DC from Mif−/−STZ to induce proliferation in ovalbumin-specific T cells. All the deficiencies observed in Mif−/−STZ were recovered by exogenous administration of recombinant Mif. These findings suggest that Mif plays a role in the molecular mechanisms of Mφ and DC activation and drives T cell responses involved in the pathology of T1DM. Therefore, Mif is a potential therapeutic target to reduce the pathology of T1DM. PMID:27699180

  15. Transcriptome profile reveals AMPA receptor dysfunction in the hippocampus of the Rsk2-knockout mice, an animal model of Coffin-Lowry syndrome.

    PubMed

    Mehmood, Tahir; Schneider, Anne; Sibille, Jérémie; Sibillec, Jérémie; Marques Pereira, Patricia; Pannetier, Solange; Ammar, Mohamed Raafet; Dembele, Doulaye; Thibault-Carpentier, Christelle; Rouach, Nathalie; Hanauer, André

    2011-03-01

    Coffin-Lowry syndrome (CLS) is a syndromic form of mental retardation caused by loss of function mutations in the X-linked RPS6KA3 gene, which encodes RSK2, a serine/threonine kinase acting in the MAPK/ERK pathway. The mouse invalidated for the Rps6ka3 (Rsk2-KO) gene displays learning and long-term spatial memory deficits. In the current study, we compared hippocampal gene expression profiles from Rsk2-KO and normal littermate mice to identify changes in molecular pathways. Differential expression was observed for 100 genes encoding proteins acting in various biological pathways, including cell growth and proliferation, cell death and higher brain function. The twofold up-regulated gene (Gria2) was of particular interest because it encodes the subunit GLUR2 of the AMPA glutamate receptor. AMPA receptors mediate most fast excitatory synaptic transmission in the central nervous system. We provide evidence that in the hippocampus of Rsk2-KO mice, expression of GLUR2 at the mRNA and at the protein levels is significantly increased, whereas basal AMPA receptor-mediated transmission in the hippocampus of Rsk2-KO mice is significantly decreased. This is the first time that such deregulations have been demonstrated in the mouse model of the Coffin-Lowry syndrome. Our findings suggest that a defect in AMPA neurotransmission and plasticity contribute to mental retardation in CLS patients.

  16. Altered Macrophage and Dendritic Cell Response in Mif-/- Mice Reveals a Role of Mif for Inflammatory-Th1 Response in Type 1 Diabetes.

    PubMed

    Sánchez-Zamora, Yuriko Itzel; Juarez-Avelar, Imelda; Vazquez-Mendoza, Alicia; Hiriart, Marcia; Rodriguez-Sosa, Miriam

    2016-01-01

    Macrophage migration inhibitory factor (Mif) is highly expressed in type 1 diabetes mellitus (T1DM). However, there is limited information about how Mif influences the activation of macrophages (Mφ) and dendritic cells (DC) in T1DM. To address this issue, we induced T1DM by administering multiple low doses of streptozotocin (STZ) to Mif-/- or wild-type (Wt) BALB/c mice. We found that Mif-/- mice treated with STZ (Mif-/-STZ) developed lower levels of hyperglycemia, inflammatory cytokines, and specific pancreatic islet antigen- (PIAg-) IgG and displayed reduced cellular infiltration into the pancreatic islets compared to Wt mice treated with STZ (WtSTZ). Moreover, Mφ and DC from Mif-/-STZ displayed lower expression of MHC-II, costimulatory molecules CD80, CD86, and CD40, Toll-like receptor- (TLR-) 2, and TLR-4 than WtSTZ. These changes were associated with a reduced capacity of Mφ and DC from Mif-/-STZ to induce proliferation in ovalbumin-specific T cells. All the deficiencies observed in Mif-/-STZ were recovered by exogenous administration of recombinant Mif. These findings suggest that Mif plays a role in the molecular mechanisms of Mφ and DC activation and drives T cell responses involved in the pathology of T1DM. Therefore, Mif is a potential therapeutic target to reduce the pathology of T1DM.

  17. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development

    PubMed Central

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-01-01

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes. PMID:27917906

  18. In vivo two-photon imaging reveals a role of progesterone in reducing axonal dieback after spinal cord injury in mice.

    PubMed

    Yang, Zhijie; Xie, Wenguang; Ju, Furong; Khan, Akbar; Zhang, Shengxiang

    2017-04-01

    Progesterone (PG) as a neuroprotective reagent has been used for the treatment of spinal cord injury (SCI) in experimental animal models. However, its effect and mechanism on axonal dieback at the early stage of SCI remain unclear. Here, we investigate the dynamics of injured axons and the effect of PG on the axonal dieback, glial response, and behavioral recovery in a mouse model of SCI. Two-photon intravital imaging combined with a simplified imaging window chamber were used to image axons in hemisected spinal cords over a period of 3 days. Repeated imaging showed that axonal dieback distance in mice treated with PG after SCI was significantly reduced than that in mice treated with vehicle after SCI (P < 0.05) at the time point of 24 h, 48 h, and 72 h after SCI. The densities of astrocytes and microglia in the SCI-vehicle treated group were significantly higher than those in mice treated with PG after SCI (P < 0.05). Real time polymerase chain reaction assay indicated that administration of PG after SCI down-regulated the expression of pro-inflammatory cytokines MCP-1, NOS2, and IL-1β (P < 0.05). PG treatment also improved the behavioral performance post injury. These findings suggested that PG exerted a neuroprotective effect by attenuating axonal dieback, reducing the accumulation of astrocytes and microglia and inhibiting the release of pro-inflammatory cytokines.

  19. Differential proteomic analysis of the pancreas of diabetic db/db mice reveals the proteins involved in the development of complications of diabetes mellitus.

    PubMed

    Pérez-Vázquez, Victoriano; Guzmán-Flores, Juan M; Mares-Álvarez, Daniela; Hernández-Ortiz, Magdalena; Macías-Cervantes, Maciste H; Ramírez-Emiliano, Joel; Encarnación-Guevara, Sergio

    2014-05-30

    Type 2 diabetes mellitus is characterized by hyperglycemia and insulin-resistance. Diabetes results from pancreatic inability to secrete the insulin needed to overcome this resistance. We analyzed the protein profile from the pancreas of ten-week old diabetic db/db and wild type mice through proteomics. Pancreatic proteins were separated in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significant changes in db/db mice respect to wild type mice were observed in 27 proteins. Twenty five proteins were identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and their interactions were analyzed using search tool for the retrieval of interacting genes/proteins (STRING) and database for annotation, visualization and integrated discovery (DAVID). Some of these proteins were Pancreatic α-amylase, Cytochrome b5, Lithostathine-1, Lithostathine-2, Chymotrypsinogen B, Peroxiredoxin-4, Aspartyl aminopeptidase, Endoplasmin, and others, which are involved in the metabolism of carbohydrates and proteins, as well as in oxidative stress, and inflammation. Remarkably, these are mostly endoplasmic reticulum proteins related to peptidase activity, i.e., they are involved in proteolysis, glucose catabolism and in the tumor necrosis factor-mediated signaling pathway. These results suggest mechanisms for insulin resistance, and the chronic inflammatory state observed in diabetes.

  20. Mirrored bilateral slow-wave cortical activity within local circuits revealed by fast bihemispheric voltage-sensitive dye imaging in anesthetized and awake mice.

    PubMed

    Mohajerani, Majid H; McVea, David A; Fingas, Matthew; Murphy, Timothy H

    2010-03-10

    Spontaneous slow-wave oscillations of neuronal membrane potential occur about once every second in the rodent cortex and may serve to shape the efficacy of evoked neuronal responses and consolidate memory during sleep. However, whether these oscillations reflect the entrainment of all cortical regions via propagating waves or whether they exhibit regional and temporal heterogeneity that reflects processing in local cortical circuits is unknown. Using voltage-sensitive dye (VSD) imaging within an adult C57BL/6J mouse cross-midline large craniotomy preparation, we recorded this depolarizing activity across most of both cortical hemispheres simultaneously in both anesthetized and quiet awake animals. Spontaneous oscillations in the VSD signal were highly synchronized between hemispheres, and acallosal I/LnJ mice indicated that synchrony depended on the corpus callosum. In both anesthetized and awake mice (recovered from anesthesia), the oscillations were not necessarily global changes in activity state but were made up of complex local patterns characterized by multiple discrete peaks that were unevenly distributed across cortex. Although the local patterns of depolarizing activity were complex and changed over tens of milliseconds, they were faithfully mirrored in both hemispheres in mice with an intact corpus callosum, to perhaps ensure parallel modification of related circuits in both hemispheres. We conclude that within global rhythms of spontaneous activity are complex events that reflect orchestrated processing within local cortical circuits.

  1. Upregulation of class I major histocompatibility complex gene expression in primary sensory neurons, satellite cells, and Schwann cells of mice in response to acute but not latent herpes simplex virus infection in vivo

    PubMed Central

    1994-01-01

    Major histocompatibility complex (MHC) deficiency is typical of almost all resident cells in normal neural tissue. However, CD8+ T cells, which recognize antigenic peptides in the context of class I MHC molecules, are known to mediate clearance of herpes simplex virus (HSV) from spinal ganglia of experimentally infected mice, leading to the hypothesis that class I expression in the peripheral nervous system must be upregulated in response to HSV infection. In addressing this hypothesis it is shown, in BALB/c (H-2d) mice, that normally deficient class I transcripts transiently accumulate in peripheral nerve Schwann cells, ganglionic satellite cells, and primary sensory neurons, indicating that in each of these cell types class I expression is regulated at the transcriptional level in vivo. Furthermore, for 3-4 wk after infection, H-2Kd/Dd antigens are expressed by satellite and Schwann cells but not neurons, suggesting additional posttranscriptional regulation of class I synthesis in neurons. Alternatively, the class I RNAs induced in neurons may not be derived from classical class I genes. Factors regulating H-2 class I expression emanate from within infected ganglia, probably from infected neurons themselves. However, induction of class I molecules was not maintained during latency, when viral gene expression in neurons is restricted to a single region within the virus repeats. These data have implications for the long-term survival of cells in HSV-infected neural tissue. PMID:8064236

  2. Mechanisms of immunity to Leishmania major infection in mice: the contribution of DNA vaccines coding for two novel sets of histones (H2A-H2B or H3-H4).

    PubMed

    Carrión, Javier

    2011-09-01

    The immune phenotype conferred by two different sets of histone genes (H2A-H2B or H3-H4) was assessed. BALB/c mice vaccinated with pcDNA3H2AH2B succumbed to progressive cutaneous leishmaniosis (CL), whereas vaccination with pcDNA3H3H4 resulted in partial resistance to Leishmania major challenge associated with the development of mixed T helper 1 (Th1)/Th2-type response and a reduction in parasite-specific Treg cells number at the site of infection. Therefore, the presence of histones H3 and H4 may be considered essential in the development of vaccine strategies against CL based on the Leishmania histones.

  3. 4-chloro-o-phenylenediamine induces a dose-related increase in G:C > T:A transversions and one major DNA adduct in the liver of Big Blue mice after 26 weeks in feed treatment.

    PubMed

    Staedtler, F; Crespo-Perez, J; Sagelsdorff, P; Steiner, S; Suter, W

    1999-11-29

    The monocyclic aromatic amine 4-chloro-o-phenylenediamine (4-C-o-PDA), a known mutagen and mouse hepatocarcinogen, was tested for its in vivo mutagenic potential in the Big Blue transgenic mouse assay system. Genomic DNA was isolated from liver tissue of control and treated animals and lacI mutants were recovered. In an initial 2-week study 4-C-o-PDA was administered daily per os to groups of male and female C57BL/6 Big Blue mice at doses of 0 and 200 mg/kg for 2 weeks (on working days) followed by a treatment free expression time of 10 days. Only a weak increase in the mutant frequencies in females was observed. In a 26-week study, where 4-C-o-PDA was given to groups of male and female Big Blue mice in feed at dietary concentrations of 0, 5,000 and 10,000 ppm, 4-C-o-PDA was found to induce a pronounced dose-dependent increase in mutant frequencies in either sex. In the present work, we analyzed the mutation spectrum by automated DNA sequencing of lacI mutants from both studies. Following the 2-week administration of 4-C-oT:A transversions in both sexes. In addition, upon 26-week treatment with 4-C-o-PDA, one major DNA adduct was detected by 33P postlabelling and subsequent multidimensional thin layer chromatography. It is concluded that 4-C-oT:A transversions after 26 weeks in feed treatment. This result indicates that the sensitivity of the Big Blue transgenic assay system, in detecting a unique chemically induced mutation spectrum, is dependent on experimental parameters, such as treatment time. The data suggest that the formation of one major DNA adduct upon 4-C-o-PDA treatment may be critical for its mutagenicity.

  4. Regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00278a Click here for additional data file.

    PubMed Central

    Enk, Viktoria M.; Baumann, Christian; Thoß, Michaela; Luzynski, Kenneth C.; Razzazi-Fazeli, Ebrahim

    2016-01-01

    Major urinary proteins (MUPs) are highly homologous proteoforms that function in binding, transporting and releasing pheromones in house mice. The main analytical challenge for studying variation in MUPs, even for state-of-the-art proteomics techniques, is their high degree of amino acid sequence homology. In this study we used unique peptides for proteoform-specific identification. We applied different search engines (ProteinPilot™ vs. PEAKS®) and protein databases (MUP database vs. SwissProt + unreviewed MUPs), and found that proteoform identification is influenced by addressing background proteins (unregulated urinary proteins, non-MUPs) during the database search. High resolution Q-TOF mass spectrometry was used to identify and precisely quantify the regulation of MUP proteoforms in male mice that were reared in standard housing and then transferred to semi-natural enclosures (within-subject design). By using a designated MUP database we were able to distinguish 19 MUP proteoforms, with A2CEK6 (a Mup11 gene product) being the most abundant based on spectral intensities. We compared three different quantification strategies based on MS1- (from IDA and SWATH™ spectra) and MS2 (SWATH™) data, and the results of these methods were correlated. Furthermore, three data normalization methods were compared and we found that increased statistical significance of fold-changes can be achieved by normalization based on urinary protein concentrations. We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs (differential regulation), e.g., A2ANT6, a Mup6 gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database. Finally, we show that 85 ± 7% of total MS intensity can be attributed to MUP-derived peptides, which supports the assumption that MUPs are the primary proteins in mouse urine. Our results provide new tools for assessing qualitative and quantitative variation of MUPs

  5. Major depression

    MedlinePlus

    Depression - major; Depression - clinical; Clinical depression; Unipolar depression; Major depressive disorder ... providers do not know the exact causes of depression. It is believed that chemical changes in the ...

  6. α-Pinene, a Major Constituent of Pine Tree Oils, Enhances Non-Rapid Eye Movement Sleep in Mice through GABAA-benzodiazepine Receptors.

    PubMed

    Yang, Hyejin; Woo, Junsung; Pae, Ae Nim; Um, Min Young; Cho, Nam-Chul; Park, Ki Duk; Yoon, Minseok; Kim, Jiyoung; Lee, C Justin; Cho, Suengmok

    2016-11-01

    α-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that α-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of α-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (-)-α-pinene shows sleep enhancing property through a direct binding to GABAA-benzodiazepine (BZD) receptors by acting as a partial modulator at the BZD binding site. The effect of (-)-α-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (-)-α-pinene was investigated by electrophysiology and molecular docking study. (-)-α-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (-)-α-pinene potentiated the GABAA receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (-)-α-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABAA-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABAA-BZD receptor. (-)-α-pinene was found to bind to aromatic residues of α1- and -γ2 subunits of GABAA-BZD receptors in the molecular model. We conclude that (-)-α-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABAA-BZD receptors and directly binding to the BZD binding site of GABAA receptor.

  7. Expression pattern of NMDA receptors reveals antiepileptic potential of apigenin 8-C-glucoside and chlorogenic acid in pilocarpine induced epileptic mice.

    PubMed

    Aseervatham, G Smilin Bell; Suryakala, U; Doulethunisha; Sundaram, S; Bose, P Chandra; Sivasudha, T

    2016-08-01

    The present study was aimed to evaluate the effect of apigenin 8-C-glucoside (Vitexin) and chlorogenic acid on epileptic mice induced by pilocarpine and explored its possible mechanisms. Intraperitonial administration of pilocarpine (85mg/kg) induced seizure in mice was assessed by behavior observations, which is significantly (p>0.05) reduced by apigenin 8-C-glucoside (AP8CG) (10mg/kg) and chlorogenic acid (CA) (5mg/kg), similar to diazepam. Seizure was accompanied by an imbalance in the levels of Gamma-aminobutyric acid (GABA) and glutamate in the pilocarpine administered group. Moreover, convulsion along with reduced acetylcholinesterase, increased monoamine oxidase and oxidative stress was observed in epileptic mice brain. AP8CG and CA significantly restored back to normal levels even at lower doses. Further, increased lipid peroxidation and nitrite content was also significantly attenuated by AP8CG and CA. However, CA was found to be more effective when compared to AP8CG. In addition, the mRNA expression of N-methyl-d-aspartate receptor (NMDAR), mGluR1 and mGlu5 was significantly (P≤0.05) inhibited by AP8CG and CA in a lower dose. The mRNA expression of GRIK1 did not differ significantly in any of the group and showed a similar pattern of expression. Our result shows that AP8CG and CA selectively inhibit NMDAR, mGluR1 and mGlu5 expression. Modification in the provoked NMDAR calcium response coupled with neuronal death. Hence, these findings underline that the polyphenolics, AP8CG and CA have exerted antiepileptic and neuroprotective activity by suppressing glutamate receptors.

  8. Assessing Interactions Between Ghsr and Mc3r Reveals a Role for AgRP in the Expression of Food Anticipatory Activity in Male Mice

    PubMed Central

    Girardet, Clemence; Mavrikaki, Maria; Southern, Mark R.; Smith, Roy G.

    2014-01-01

    The stomach hormone ghrelin and hypothalamic melanocortin neurons belong to a gut-brain circuit controlling appetite and metabolic homeostasis. Mice lacking melanocortin-3 receptor (Mc3rKO) or growth hormone secretagogue receptor (GhsrKO) genes exhibit attenuated food anticipatory activity (FAA), a rise in locomotor activity anticipating mealtime, suggesting common circuitry regulating anticipatory responses to nutrient loading. To investigate the interaction between Ghsrs and Mc3rs, we compared food anticipatory responses in GhsrKO, Mc3rKO, and double Ghsr;Mc3r knockout (DKO) mice subjected to a hypocaloric restricted feeding (RF) protocol in constant dark or 12-hour light, 12-hour dark settings. DKO are viable, exhibiting no overt behavioral or metabolic phenotypes in ad libitum or fasting conditions. FAA was initially attenuated in all mutant strains in constant darkness. However, GhsrKO eventually exhibited a robust food anticipatory response, suggesting compensation. Mc3rKO and DKO did not compensate, indicating a continued requirement for Mc3rs in maintaining the expression of FAA in situations of RF. Abnormal regulation of hypothalamic agouti-related peptide/neuropeptide Y (AgRP/Npy) neurons previously observed during fasting may contribute to attenuated FAA in Mc3rKO. AgRP and Npy expression measured 1 hour before food presentation correlated positively with FAA. Absence of Mc3rs (but not Ghsrs) was associated with lower AgRP/Npy expression, suggesting attenuated responses to signals of negative energy balance. These observations support the importance of Mc3rs as modulators of anticipatory responses to feeding, with mice able to compensate for loss of Ghsrs. The behavioral deficits of Mc3rKO displayed during RF may be partially explained by reduced hunger sensations owing to abnormal regulation of orexigenic AgRP/Npy neurons. PMID:25211592

  9. Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice.

    PubMed

    Swenson, E Scott; Guest, Ian; Ilic, Zoran; Mazzeo-Helgevold, Maria; Lizardi, Pablo; Hardiman, Camille; Sell, Stewart; Krause, Diane S

    2008-07-01

    The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

  10. Timed conditional null of connexin26 in mice reveals temporary requirements of connexin26 in key cochlear developmental events before the onset of hearing.

    PubMed

    Chang, Qing; Tang, Wenxue; Kim, Yeunjung; Lin, Xi

    2015-01-01

    Mutations in the Gjb2 gene, which encodes a gap junction protein connexin26 (Cx26), are the most prevalent form of hereditary deafness in humans and represent about half of non-syndromic congenital deafness cases in many ethnic populations. Cochlear potassium (K+) recycling in mature cochlea is required for normal hearing. It is thought that gap junctions are essential for K+ recycling and that Gjb2 mutations cause Gjb2-associated deafness by disrupting K+ recycling in mature cochlea. Here we present evidence showing that Gjb2 is required for normal development of the neonatal organ of Corti prior to the onset of the hearing in mice. In the conditional Gjb2 null (cCx26 null) mice, ribbon synapses in inner hair cells remained poorly developed, the afferent type I fibers failed to finish the refinement process to form convergent innervation to individual inner hair cells. The spontaneous depolarizing activities in the supporting cells, which normally diminish in the wild type cochleae after postnatal day 8 (P8), remained strong in the cochlea after P8 in the mutant mice. Furthermore, the deafness phenotype was readily generated only if the Cx26 expression in the organ of Corti was significantly reduced before P6. Similar amount of Cx26 reduction in more mature cochleae had a much weaker effect in damaging the hearing sensitivity. Our findings indicated that Cx26 plays essential roles in the maturation process of the organ of Corti prior to the establishment of high K+ in the endolymph and the onset of hearing. These results suggest that successful treatment of Cx26 deafness requires early intervention before the cochlea fully matures.

  11. Intra-islet lesions and lobular variations in β-cell mass expansion in ob/ob mice revealed by 3D imaging of intact pancreas

    PubMed Central

    Parween, Saba; Kostromina, Elena; Nord, Christoffer; Eriksson, Maria; Lindström, Per; Ahlgren, Ulf

    2016-01-01

    The leptin deficient ob/ob mouse is a widely used model for studies on initial aspects of metabolic disturbances leading to type 2 diabetes, including insulin resistance and obesity. Although it is generally accepted that ob/ob mice display a dramatic increase in β-cell mass to compensate for increased insulin demand, the spatial and quantitative dynamics of β-cell mass distribution in this model has not been assessed by modern optical 3D imaging techniques. We applied optical projection tomography and ultramicroscopy imaging to extract information about individual islet β-cell volumes throughout the volume of ob/ob pancreas between 4 and 52 weeks of age. Our data show that cystic lesions constitute a significant volume of the hyperplastic ob/ob islets. We propose that these lesions are formed by a mechanism involving extravasation of red blood cells/plasma due to increased islet vessel blood flow and vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of the characteristics of β-cell expansion in ob/ob mice presented in this report may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function. PMID:27713548

  12. Multimodal coherent anti-Stokes Raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice

    NASA Astrophysics Data System (ADS)

    Imitola, Jaime; Côté, Daniel; Rasmussen, Stine; Xie, X. Sunney; Liu, Yingru; Chitnis, Tanuja; Sidman, Richard L.; Lin, Charles. P.; Khoury, Samia J.

    2011-02-01

    Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH2 vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX3CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration.

  13. Multimodal coherent anti-Stokes Raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice

    PubMed Central

    Imitola, Jaime; Côté, Daniel; Rasmussen, Stine; Xie, X. Sunney; Liu, Yingru; Chitnis, Tanuja; Sidman, Richard L.; Lin, Charles. P.; Khoury, Samia J.

    2011-01-01

    Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH2 vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX3CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration. PMID:21361672

  14. Syrtis Major

    NASA Technical Reports Server (NTRS)

    2002-01-01

    , scientists were fascinated with Syrtis Major because this dark region varied so much through the seasons and years. Some people thought it might be a changing sea, and others thought it might be vegetation. Early spacecraft like Mariner and Viking revealed for the first time that the changes were caused by the wind blowing dust and sand across the surface. What we can see in this image is exactly that: evidence of a lot of wind action. Bright dust patches streak across this image, formed through wind interference from craters and other landforms. These wispy, bright streaks are spread on the surface by a vigorous, east-west wind that kicked up huge dust storms, scattering the fine particles of sand and dust in an almost etherial pattern. The bright streaks in the top part of the image might have formed in a slightly different way, because there is no landform standing in the wind's way. Beneath the bright surface dust are raised ridges that mark the edges of earlier lava flows from Nili Patera, a Martian 'caldera.' A caldera is a collapsed, bowl-shaped depression at the top of a volcano cone. Can you imagine how Christian Huygens would feel if he lived today and could see all of this knowledge unfold? Or how it would feel to be the first person to stand in this dark volcanic and cratered region, knowing how many discovers had paved the way to that moment? Yes, exploration lives!

  15. Syrtis Major

    NASA Technical Reports Server (NTRS)

    2002-01-01

    (Released 6 June 2002) The Science This image, located near the equator and 288W (72E), is near the southern edge of a low, broad volcanic feature called Syrtis Major. A close look at this image reveals a wrinkly texture that indicates a very rough surface that is associated with the lava flows that cover this region. On a larger scale, there are numerous bright streaks that trail topographic features such as craters. These bright streaks are in the wind shadows of the craters where dust that settles onto the surface is not as easily scoured away. It is important to note that these streaks are only bright in a relative sense to the surrounding image. Syrtis Major is one of the darkest regions on Mars and it is as dark as fresh basalt flows or dunes are on Earth. The Story Cool! It almost looks as if nature has 'painted' comets on the surface of Mars, using craters as comet cores and dust as streaky tails. Of course, that's just an illusion. As in many areas of Mars, the wind is behind the creation of such fantastic landforms. The natural phenomenon seen here gives this particular surface of Mars a very dynamic, fast-moving, almost luminous 'cosmic personality.' The bright, powdery-looking streaks of dust are in the 'wind shadows' of craters, where dust that settles onto the surface is not as easily scoured away. That's because the wind moves across the land in a particular direction, and a raised surface like the rim of a crater 'protects' dust from being completely blown away on the other side. The raised landforms basically act as a buffer. From the streaks seen above, you can tell the wind was blowing in a northeast to southwest direction. Why are the streaks so bright? Because they contrast with the really dark underlying terrain in this volcanic area of Mars. Syrtis Major is one of the darkest regions on Mars because it is made of basalt. Basalt is typically dark gray or black, and forms when a certain type of molten lava cools. The meaning of the word basalt

  16. Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response

    PubMed Central

    2010-01-01

    Background Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data. Results A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results. Conclusions Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against

  17. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

    PubMed

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-03-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

  18. Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice

    PubMed Central

    Lim, Joohyun; Burclaff, Joseph; He, Guangxu; Mills, Jason C; Long, Fanxin

    2017-01-01

    Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmp1-Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmp1 gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmp1-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmp1-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmp1-Cre in the gastrointestinal mesenchyme, deletion of Bmpr1a with Dmp1-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmp1-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types. PMID:28163952

  19. Social interaction and cocaine conditioning in mice increase spontaneous spike frequency in the nucleus accumbens or septal nuclei as revealed by multielectrode array recordings.

    PubMed

    Kummer, Kai K; El Rawas, Rana; Kress, Michaela; Saria, Alois; Zernig, Gerald

    2015-01-01

    Both cocaine and social interaction place preference conditioning lead to increased neuronal expression of the immediate early gene EGR1 in the nucleus accumbens, a central region of the reward pathway, suggesting that both drug and natural rewards may be processed in similar brain regions. In order to gain novel insights into the intrinsic in vitro electrical activity of the nucleus accumbens and adjacent brain regions and to explore the effects of reward conditioning on network activity, we performed multielectrode array recordings of spontaneous firing in acute brain slices of mice conditioned to either cocaine or social interaction place preference. Cocaine conditioning increased the spike frequency of neurons in the septal nuclei, whereas social interaction conditioning increased the spike frequency in the nucleus accumbens compared to saline control animals. In addition, social interaction conditioning decreased the amount of active neuron clusters in the nucleus accumbens. Our findings suggest that place preference conditioning for both drug and natural rewards may induce persistent changes in neuronal network activity in the nucleus accumbens and the septum that are still preserved in acute slice preparations.

  20. Embryonic mutation as a possible cause of in utero carcinogenesis in mice revealed by postnatal treatment with 12-O-tetradecanoylphorbol-13-acetate

    SciTech Connect

    Nomura, T.; Nakajima, H.; Hatanaka, T.; Kinuta, M.; Hongyo, T. )

    1990-04-01

    Although in utero irradiation at early stages induced a high incidence of somatic mutations at coat color genes in the embryos of a specified tester strain (PT x HT F1) of mice, it was not carcinogenic by itself. However, in utero-irradiated animals did develop skin tumors and hepatomas (but not leukemias) by the postnatal administration of 12-O-tetradecanoylphorbol-13-acetate. The incidence of both tumors and embryonic mutations increased with in utero doses of X-rays. Furthermore, a large reduction of tumor incidence, about 80%, was observed by low-dose-rate irradiation, similar to the 75% reduction in spot size found for embryonic mutations. The tumor nodule size was also dramatically reduced by low-dose-rate irradiation. Consequently, the induced incidence and size of tumors produced by 12-O-tetradecanoylphorbol-13-acetate treatment parallel those which are observed for coat color mutations as expected, because somatic mutations observed in the pigment cells must similarly occur in embryonic cells of other organs. The larger the clone of mutant cells, the greater their chance of becoming tumorigenic by 12-O-tetradecanoylphorbol-13-acetate posttreatment. These results strongly support the recent epidemiological survey showing that adult types of cancers, but not leukemias, are increasing in the atomic bomb survivors exposed in utero, since humans are continuously exposed to a variety of cancer-promoting agents in contrast to experimental animals reared without such exposures.

  1. RNA sequencing reveals differential expression of mitochondrial and oxidation reduction genes in the long-lived naked mole-rat when compared to mice.

    PubMed

    Yu, Chuanfei; Li, Yang; Holmes, Andrew; Szafranski, Karol; Faulkes, Chris G; Coen, Clive W; Buffenstein, Rochelle; Platzer, Matthias; de Magalhães, João Pedro; Church, George M

    2011-01-01

    The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly 300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein. Also of interest are the protease inhibitor, alpha2-macroglobulin (A2m), and the mitochondrial complex II subunit Sdhc, both ageing-related genes found strongly over-expressed in the naked mole-rat. These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat's fascinating characteristics.

  2. Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons

    PubMed Central

    Encinas, Mario; Rozen, Esteban J.; Dolcet, Xavier; Jain, Sanjay; Comella, Joan X.; Milbrandt, Jeffrey; Johnson, Eugene M.

    2009-01-01

    We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways. PMID:18497757

  3. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

    PubMed Central

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-01-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

  4. Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice

    PubMed Central

    Macedo, Claudia; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Passos, Geraldo A.

    2015-01-01

    In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4+ and CD8+ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3+ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs. PMID:26606254

  5. Bone marrow transplantation across major histocompatibility barriers. V. Protection of mice from lethal graft-vs. -host disease by pretreatment of donor cells with monoclonal anti-Thy-1. 2 coupled to the toxin ricin

    SciTech Connect

    Vallera, D.A.; Youle, R.J.; Neville, D.M. Jr.; Kersey, J.H.

    1982-03-01

    A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and thus prevent graft-vs.-host disease (GVHD). The method utilizes a monoclonal antibody directed at the Thy-1.2 antigen but is complement independent. To make anti-Thy-1.2 toxic, the antibody is covalently linked to the toxin ricin. Ricin ordinarily binds, enters, and kills cells through receptors containing galactose. The hybrid protein, anti-Thy-1.2-ricin, can enter and kill cells via the Thy-1.2 receptor. In the presence of lactose the usual entry route for ricin is largely blocked and the hybrid is shown to be a highly selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes. We have used a model of severe and fatal GVHD where BALB/c splenocytes and bone marrow cells are given to irradiated C57BL/6 recipients. Over 90% of these mice die by day 70, exhibiting signs of GVHD. When donor cells are pretreated with 0.5 microgram/ml of anti-Thy-1.2-ricin plus 200 mM lactose before injection, 10 of 11 animals survive through day 70 without signs of GVHD. These studies demonstrate that ricin linked to monoclonal antibodies may have utility related to the prevention of GVHD in human bone marrow transplantation.

  6. Major Links.

    ERIC Educational Resources Information Center

    Henderson, Tona

    1995-01-01

    Provides electronic mail addresses for resources and discussion groups related to the following academic majors: art, biology, business, chemistry, computer science, economics, health sciences, history, literature, math, music, philosophy, political science, psychology, sociology, and theater. (AEF)

  7. Variation of pathways and network profiles reveals the differential pharmacological mechanisms of each effective component to treat middle cerebral artery ischemia-reperfusion mice

    PubMed Central

    Dang, HaiXia; Li, KangNing; Yu, YaNan; Zhang, YingYing; Liu, Jun; Wang, PengQian; Li, Bing; Wang, HaiNan; Li, Haixia; Wang, YongYan

    2015-01-01

    Using a system pharmacology strategy, this study evaluated the unique pharmacological characteristics of three different neuroprotective compounds for the treatment of cerebral ischemia-reperfusion. A microarray including 374 brain ischemia-related genes was used to identify the differentially expressed genes among five treatment groups: baicalin, jasminoidin, ursodeoxycholic acid, sham, and vehicle, and MetaCore analysis software was applied to identify the significantly altered pathways, processes and interaction network parameters. At pathway level, 46, 25, and 31 pathways were activated in the baicalin, jasminoidin, and ursodeoxycholic acid groups, respectively. Thirteen pathways mainly related with apoptosis and development were commonly altered in the three groups. Additionally, baicalin also targeted pathways related with development, neurophysiologic process and cytoskeleton remodeling, while jasminoidin targeted pathways related with cell cycle and ursodeoxycholic acid targeted those related with apoptosis and development. At process level, three processes were commonly regulated by the three groups in the top 10 processes. Further interaction network analysis revealed that baicalin, jasminoidin, and ursodeoxycholic acid displayed unique features either on network topological parameters or network structure. Additional overlapping analysis demonstrated that compared with ursodeoxycholic acid, the pharmacological mechanism of baicalin was more similar with that of jasminoidin in treating brain ischemia. The data presented in this study may contribute toward the understanding of the common and differential pharmacological mechanisms of these three compounds. PMID:26168995

  8. Pharmacokinetics and metabolism of midazolam in chimeric mice with humanised livers.

    PubMed

    Samuelsson, Kristin; Pickup, Kathryn; Sarda, Sunil; Swales, John G; Morikawa, Yoshio; Schulz-Utermoehl, Timothy; Hutchison, Michael; Wilson, Ian D

    2012-11-01

    The pharmacokinetics and biotransformation of midazolam were investigated following single oral doses of 0.1, 1 and 10 mg/kg to chimeric mice with humanised livers (PXB mice) and to severe combined immunodeficient (SCID) mice used as controls. Pharmacokinetic analysis, on whole blood, revealed rapid absorption of the administered midazolam with a higher C(max) in PXB compared to SCID. The exposure to 1'-hydroxymidazolam was approximately 14-fold greater than to midazolam in the SCID mice and close to equivalent in the PXB mice. The metabolism of midazolam in SCID mice was faster than in the PXB mice such that pharmacokinetic data for midazolam in SCID mice could not be generated from the lowest dose in these animals. Both oxidative and conjugative metabolic pathways were identified in the PXB mice. All the major circulating metabolites observed in humans; 1'-hydroxymidazolam, 4'-hydroxymidazolam, 1',4'-dihydroxymidazolam and 1'-hydroxymidazolam glucuronide, were detected in the blood of PXB mice. However, 4'-hydroxymidazolam and the 1'-hydroxymidazolam glucuronide were not detected in blood samples obtained from SCID mice. The midazolam metabolite profile in the PXB mouse was similar to that previously reported for human suggesting that the PXB mouse model can provide a model system for predicting circulating human metabolites.

  9. Major depression.

    PubMed

    Bentley, Susan M; Pagalilauan, Genevieve L; Simpson, Scott A

    2014-09-01

    Major depression is a common, disabling condition seen frequently in primary care practices. Non-psychiatrist ambulatory providers are increasingly responsible for diagnosing, and primarily managing patients suffering from major depressive disorder (MDD). The goal of this review is to help primary care providers to understand the natural history of MDD, identify practical tools for screening, and a thoughtful approach to management. Clinically challenging topics like co-morbid conditions, treatment resistant depression and pharmacotherapy selection with consideration to side effects and medication interactions, are also covered.

  10. Major Andre

    ERIC Educational Resources Information Center

    Henisch, B. A.; Henisch, H. K.

    1976-01-01

    If most Revolutionary era people seem two-dimensional their lives simpler to understand than ours, it may be only that history, with the benefit of hindsight, clarifies. Examines a profile of Major John Andre, the British liaison officer in Benedict Arnold's plan to surrender West Point, as both hero and villain to show the complexity of early…

  11. Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes.

    PubMed

    Lee, H M; Wu, W; Wysoczynski, M; Liu, R; Zuba-Surma, E K; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2009-11-01

    We reported that complement cascade (CC) becomes activated in bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization of hematopoietic stem/progenitor cells (HSPCs) and showed that, although third CC component (C3)-deficient mice are easy mobilizers, fifth CC component (C5)-deficient mice mobilize very poorly. To explain this, we postulated that activation/cleavage of CC releases C3a and C5a anaphylatoxins that differently regulate mobilization. Accordingly, C3a, by enhancing responsiveness of HSPCs to decreasing concentrations of stromal-derived growth factor-1 (SDF-1) in BM, prevents mobilization and promotes their BM retention. Therefore, in this study, we focused on the mobilization-enhancing role of C5a. We found that C5a receptor (C5aR) is not expressed on the surface of HSPCs, and that C5a-mediated promobilization effects are mediated by stimulation of granulocytes. Overall, our data support the following model. First C5aR(+) granulocytes are chemoattracted by plasma C5 cleavage fragments, being the first wave of cells leaving BM. This facilitates a subsequent egress of HSPCs. In the next step, after leaving BM, granulocytes undergo degranulation in response to plasma C5a and secrete some cationic peptides (cathelicidin, beta-defensin) that, as shown here for the first time, highly enhance the responsiveness of HSPCs to plasma SDF-1 gradient. In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization, in which C5 cleavage fragments through granulocytes orchestrate this process.

  12. Adaptation of maize to temperate climates: mid-density genome-wide association genetics and diversity patterns reveal key genomic regions, with a major contribution of the Vgt2 (ZCN8) locus.

    PubMed

    Bouchet, Sophie; Servin, Bertrand; Bertin, Pascal; Madur, Delphine; Combes, Valérie; Dumas, Fabrice; Brunel, Dominique; Laborde, Jacques; Charcosset, Alain; Nicolas, Stéphane

    2013-01-01

    The migration of maize from tropical to temperate climates was accompanied by a dramatic evolution in flowering time. To gain insight into the genetic architecture of this adaptive trait, we conducted a 50K SNP-based genome-wide association and diversity investigation on a panel of tropical and temperate American and European representatives. Eighteen genomic regions were associated with flowering time. The number of early alleles cumulated along these regions was highly correlated with flowering time. Polymorphism in the vicinity of the ZCN8 gene, which is the closest maize homologue to Arabidopsis major flowering time (FT) gene, had the strongest effect. This polymorphism is in the vicinity of the causal factor of Vgt2 QTL. Diversity was lower, whereas differentiation and LD were higher for associated loci compared to the rest of the genome, which is consistent with selection acting on flowering time during maize migration. Selection tests also revealed supplementary loci that were highly differentiated among groups and not associated with flowering time in our panel, whereas they were in other linkage-based studies. This suggests that allele fixation led to a lack of statistical power when structure and relatedness were taken into account in a linear mixed model. Complementary designs and analysis methods are necessary to unravel the architecture of complex traits. Based on linkage disequilibrium (LD) estimates corrected for population structure, we concluded that the number of SNPs genotyped should be at least doubled to capture all QTLs contributing to the genetic architecture of polygenic traits in this panel. These results show that maize flowering time is controlled by numerous QTLs of small additive effect and that strong polygenic selection occurred under cool climatic conditions. They should contribute to more efficient genomic predictions of flowering time and facilitate the dissemination of diverse maize genetic resources under a wide range of

  13. Adaptation of Maize to Temperate Climates: Mid-Density Genome-Wide Association Genetics and Diversity Patterns Reveal Key Genomic Regions, with a Major Contribution of the Vgt2 (ZCN8) Locus

    PubMed Central

    Bouchet, Sophie; Servin, Bertrand; Bertin, Pascal; Madur, Delphine; Combes, Valérie; Dumas, Fabrice; Brunel, Dominique; Laborde, Jacques; Charcosset, Alain; Nicolas, Stéphane

    2013-01-01

    The migration of maize from tropical to temperate climates was accompanied by a dramatic evolution in flowering time. To gain insight into the genetic architecture of this adaptive trait, we conducted a 50K SNP-based genome-wide association and diversity investigation on a panel of tropical and temperate American and European representatives. Eighteen genomic regions were associated with flowering time. The number of early alleles cumulated along these regions was highly correlated with flowering time. Polymorphism in the vicinity of the ZCN8 gene, which is the closest maize homologue to Arabidopsis major flowering time (FT) gene, had the strongest effect. This polymorphism is in the vicinity of the causal factor of Vgt2 QTL. Diversity was lower, whereas differentiation and LD were higher for associated loci compared to the rest of the genome, which is consistent with selection acting on flowering time during maize migration. Selection tests also revealed supplementary loci that were highly differentiated among groups and not associated with flowering time in our panel, whereas they were in other linkage-based studies. This suggests that allele fixation led to a lack of statistical power when structure and relatedness were taken into account in a linear mixed model. Complementary designs and analysis methods are necessary to unravel the architecture of complex traits. Based on linkage disequilibrium (LD) estimates corrected for population structure, we concluded that the number of SNPs genotyped should be at least doubled to capture all QTLs contributing to the genetic architecture of polygenic traits in this panel. These results show that maize flowering time is controlled by numerous QTLs of small additive effect and that strong polygenic selection occurred under cool climatic conditions. They should contribute to more efficient genomic predictions of flowering time and facilitate the dissemination of diverse maize genetic resources under a wide range of

  14. Transcriptomic comparison of cyanotoxin variants in a human intestinal model revealed major differences in oxidative stress response: effects of MC-RR and MC-LR on Caco-2 cells.

    PubMed

    Zeller, Perrine; Quenault, Hélène; Huguet, Antoine; Blanchard, Yannick; Fessard, Valérie

    2012-08-01

    Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria. Their structure includes two variable amino acids (AA) giving rise to more than 90 MC variants, however most of the studies to date have focused on the most toxic variant: microcystin LR (MC-LR). Ingestion is the major route of human exposure to MCs and several in vivo studies have demonstrated macroscopic effects on the gastro-intestinal tract. However, little information exists concerning the pathways affected by MC variants on intestinal cells. In the current study, we have investigated the effects of MC-RR and MC-LR on the human intestinal cell line Caco-2 using a non-selective method and compared their response at the pangenomic scale. The cells were incubated for 4h or 24h with a range of non-toxic concentrations of MC-RR or MC-LR. Minimal effects were observed after short term exposures (4h) to either MC variant. In contrast, dose dependent modulations of gene transcription levels were observed with MC-RR and MC-LR after 24h. The transcriptomic profiles induced by MC-RR were quite similar to those induced by MC-LR, suggestive of a largely common mechanism of toxicity. However, changes in total gene expression were more pronounced following exposure to MC-LR compared to MC-RR, as revealed by functional annotation. MC-LR affected two principal pathways, the oxidative stress response and cell cycle regulation, which did not elicit significant alteration following MC-RR exposure. This work is the first comparative description of the effects of MC-LR and MC-RR in a human intestinal cell model at the pangenomic scale. It has allowed us to propose differences in the mechanism of toxicity for MC-RR and MC-LR. These results illustrate that taking into account the toxicity of MC variants remains a key point for risk assessment.

  15. RhoA mediates defective stem cell function and heterotopic ossification in dystrophic muscle of mice.

    PubMed

    Mu, Xiaodong; Usas, Arvydas; Tang, Ying; Lu, Aiping; Wang, Bing; Weiss, Kurt; Huard, Johnny

    2013-09-01

    Heterotopic ossification (HO) and fatty infiltration (FI) often occur in diseased skeletal muscle and have been previously described in various animal models of Duchenne muscular dystrophy (DMD); however, the pathological mechanisms remain largely unknown. Dystrophin-deficient mdx mice and dystrophin/utrophin double-knockout (dKO) mice are mouse models of DMD; however, mdx mice display a strong muscle regeneration capacity, while dKO mice exhibit a much more severe phenotype, which is similar to patients with DMD. Our results revealed that more extensive HO, but not FI, occurred in the skeletal muscle of dKO mice versus mdx mice, and RhoA activation specifically occurred at the sites of HO. Moreover, the gene expression of RhoA, BMPs, and several inflammatory factors were significantly up-regulated in muscle stem cells isolated from dKO mice; while inactivation of RhoA in the cells with RhoA/ROCK inhibitor Y-27632 led to reduced osteogenic potential and improved myogenic potential. Finally, inactivation of RhoA signaling in the dKO mice with Y-27632 improved muscle regeneration and reduced the expression of BMPs, inflammation, HO, and intramyocellular lipid accumulation in both skeletal and cardiac muscle. Our results revealed that RhoA represents a major molecular switch in the regulation of HO and muscle regeneration in dystrophic skeletal muscle of mice.

  16. Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

    PubMed

    Simbolo, Michele; Mafficini, Andrea; Sikora, Katarzyna O; Fassan, Matteo; Barbi, Stefano; Corbo, Vincenzo; Mastracci, Luca; Rusev, Borislav; Grillo, Federica; Vicentini, Caterina; Ferrara, Roberto; Pilotto, Sara; Davini, Federico; Pelosi, Giuseppe; Lawlor, Rita T; Chilosi, Marco; Tortora, Giampaolo; Bria, Emilio; Fontanini, Gabriella; Volante, Marco; Scarpa, Aldo

    2017-03-01

    Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-covera