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Sample records for mice reveals major

  1. The Majority of Resorptions in Old Mice Are Euploid

    PubMed Central

    Tao, Yong; Liu, X. Johné

    2015-01-01

    Chromosomal abnormality is a leading cause of aging-related infertility, spontaneous abortion and congenital birth defects in humans. Karyotype analyses of spontaneously aborted human fetuses reveal high proportions (~50%) being chromosomal abnormal with the majority being trisomies of various chromosomes. As a model organism, mice are widely used for studies of reproduction and reproductive aging. Like older women, older mice exhibit high incidences of early embryo death. However, it is not known if aneuploidy is prevalent amongst resorptions in older mice. We have karyotyped 65 retarded/resorbed fetuses in 10-month-old C57BL/6 mice, and found that 55 (84.6%±8.8%, with 95% confidence) were euploid. Similarly, of 40 such fetuses from 17 month-old C57BL/6 mice, we found 38 (95±7%, with 95% confidence 95%) being euploid. Therefore, aneuploidy is not a leading cause of embryo death in older mice. PMID:26636341

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

    PubMed Central

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

    2011-01-01

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

  3. Leishmania tropica major in mice: vaccination against cutaneous leishmaniasis in mice of high genetic susceptibility.

    PubMed

    Mitchell, G F; Handman, E

    1983-02-01

    BALB/c and BALB/c.H-2b mice are genetically susceptible to development of persistent and severe disease following cutaneous injection of promastigotes of the protozoan parasite, Leishmania tropica major, whereas C57BL/6 are relatively resistant. Resistance in C57BL/6 can be further increased by intraperitoneal injection of living, but not killed, promastigotes prior to cutaneous challenge. Severely diseased BALB/c mice can show resistance to development of a second cutaneous lesion but apparently only in the advanced stages of systemic life-threatening disease. A striking level of resistance to persistent disease has been demonstrated in BALB/c.H-2b mice pre-injected with frozen and thawed L. t. major-infected macrophages of the continuous macrophage cell line IC-21 (H-2b) together with Corynebacterium parvum. No resistance is seen in recipients of either C. parvum or the crude antigen mixture alone. Protection is afforded by intraperitoneal and not subcutaneous injection of crude antigen plus adjuvant. In these vaccination studies all evidence points to the infected macrophage as most appropriate source of 'host-protective' antigens as well as being the most likely target of host-protective immunity. Resistance is expressed in vaccinated mice as minimal signs of cutaneous disease and rapid resolution of any small lesions which do develop. Frozen and thawed promastigotes plus C. parvum will not induce resistance to persistent disease in BALB/c.H-2b mice and preincubation of promastigotes with sera from resistant vaccinated mice does not influence their capacity to cause cutaneous disease. The results provide baseline data for vaccination attempts in genetically susceptible hosts using isolated L. t. major antigens (and, in particular, infected macrophage antigens) and highlight the utility of the intraperitoneal route of injection and the use of the therapeutic biological, C. parvum, as an adjuvant in such studies. PMID:6870673

  4. Major histocompatibility complex heterozygosity reduces fitness in experimentally infected mice.

    PubMed

    Ilmonen, Petteri; Penn, Dustin J; Damjanovich, Kristy; Morrison, Linda; Ghotbi, Laleh; Potts, Wayne K

    2007-08-01

    It is often suggested that heterozygosity at major histocompatibility complex (MHC) loci confers enhanced resistance to infectious diseases (heterozygote advantage, HA, hypothesis), and overdominant selection should contribute to the evolution of these highly polymorphic genes. The evidence for the HA hypothesis is mixed and mainly from laboratory studies on inbred congenic mice, leaving the importance of MHC heterozygosity for natural populations unclear. We tested the HA hypothesis by infecting mice, produced by crossbreeding congenic C57BL/10 with wild ones, with different strains of Salmonella, both in laboratory and in large population enclosures. In the laboratory, we found that MHC influenced resistance, despite interacting wild-derived background loci. Surprisingly, resistance was mostly recessive rather than dominant, unlike in most inbred mouse strains, and it was never overdominant. In the enclosures, heterozygotes did not show better resistance, survival, or reproductive success compared to homozygotes. On the contrary, infected heterozygous females produced significantly fewer pups than homozygotes. Our results show that MHC effects are not masked on an outbred genetic background, and that MHC heterozygosity provides no immunological benefits when resistance is recessive, and can actually reduce fitness. These findings challenge the HA hypothesis and emphasize the need for studies on wild, genetically diverse species. PMID:17603099

  5. 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

  6. Multigene phylogenetics reveals temporal diversification of major African malaria vectors.

    PubMed

    Kamali, Maryam; Marek, Paul E; Peery, Ashley; Antonio-Nkondjio, Christophe; Ndo, Cyrille; Tu, Zhijian; Simard, Frederic; Sharakhov, Igor V

    2014-01-01

    The major vectors of malaria in sub-Saharan Africa belong to subgenus Cellia. Yet, phylogenetic relationships and temporal diversification among African mosquito species have not been unambiguously determined. Knowledge about vector evolutionary history is crucial for correct interpretation of genetic changes identified through comparative genomics analyses. In this study, we estimated a molecular phylogeny using 49 gene sequences for the African malaria vectors An. gambiae, An. funestus, An. nili, the Asian malaria mosquito An. stephensi, and the outgroup species Culex quinquefasciatus and Aedes aegypti. To infer the phylogeny, we identified orthologous sequences uniformly distributed approximately every 5 Mb in the five chromosomal arms. The sequences were aligned and the phylogenetic trees were inferred using maximum likelihood and neighbor-joining methods. Bayesian molecular dating using a relaxed log normal model was used to infer divergence times. Trees from individual genes agreed with each other, placing An. nili as a basal clade that diversified from the studied malaria mosquito species 47.6 million years ago (mya). Other African malaria vectors originated more recently, and independently acquired traits related to vectorial capacity. The lineage leading to An. gambiae diverged 30.4 mya, while the African vector An. funestus and the Asian vector An. stephensi were the most closely related sister taxa that split 20.8 mya. These results were supported by consistently high bootstrap values in concatenated phylogenetic trees generated individually for each chromosomal arm. Genome-wide multigene phylogenetic analysis is a useful approach for discerning historic relationships among malaria vectors, providing a framework for the correct interpretation of genomic changes across species, and comprehending the evolutionary origins of this ubiquitous and deadly insect-borne disease. PMID:24705448

  7. Hepatic cytochrome P450s play a major role in monocrotaline-induced renal toxicity in mice

    PubMed Central

    Yao, Jun; Li, Cheng-gang; Gong, Li-kun; Feng, Chen-chen; Li, Chun-zhu; Gao, Man; Luan, Yang; Qi, Xin-ming; Ren, Jin

    2014-01-01

    Aim: Monocrotaline (MCT) in plants of the genus Crotalaria induces significant toxicity in multiple organs including the liver, lung and kidney. Metabolic activation of MCT is required for MCT-induced toxicity. In this study, we attempted to determine whether the toxicity of MCT in kidney was a consequence of the metabolic activation of MCT in the liver. Methods: Liver-specific cytochrome P450 reductase-null (Null) mice, wild-type (WT) mice and CYP3A inhibitor ketoconazole-pretreated WT (KET-WT) mice were examined. The mice were injected with MCT (300, 400, or 500 mg/kg, ip), and hepatotoxicity and nephrotoxicity were examined 24 h after MCT treatment. The levels of MCT and its metabolites in the blood, liver, lung, kidney and bile were determined using LC-MS analysis. Results: Treatment of WT mice with MCT increased the serum levels of alanine aminotransferase, hyaluronic acid, urea nitrogen and creatinine in a dose-dependent manner. Histological examination revealed that MCT (500 mg/kg) caused severe liver injury and moderate kidney injury. In contrast, these pathological abnormalities were absent in Null and KET-WT mice. After injection of MCT (400 and 500 mg/kg), the plasma, liver, kidney and lung of WT mice had significantly lower MCT levels and much higher N-oxide metabolites contents in compared with those of Null and KET-WT mice. Furthermore, WT mice had considerably higher levels of tissue-bound pyrroles and bile GSH-conjugated MCT metabolites compared with Null and KET-WT mice. Conclusion: Cytochrome P450s in mouse liver play a major role in the metabolic activation of MCT and thus contribute to MCT-induced renal toxicity. PMID:24362331

  8. 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. PMID:21225450

  9. Positional cloning of the major quantitative trait locus underlying lung tumor susceptibility in mice

    PubMed Central

    Zhang, Zhongqiu; Futamura, Manabu; Vikis, Haris G.; Wang, Min; Li, Jie; Wang, Yian; Guan, Kun-Liang; You, Ming

    2003-01-01

    Pulmonary adenoma susceptibility 1 (Pas1), located on chromosome 6, is the major locus affecting inherited predisposition to lung tumor development in mice. We have fine mapped the Pas1 locus to a region of ≈0.5 megabases by using congenic strains of mice, constructed by placing the Pas1 region of chromosome 6 from A/J mice onto the genetic background of C57BL/6J mice. Systematic characterization of Pas1 candidates establishes the Las1 (lung adenoma susceptibility 1) and Kras2 (Kirsten rat sarcoma oncogene 2) genes as primary candidates for the Pas1 locus. Clearly, Kras2 affects lung tumor progression only, and Las1 is likely to affect lung tumor multiplicity. PMID:14583591

  10. Otoancorin Knockout Mice Reveal Inertia is the Force for Hearing

    NASA Astrophysics Data System (ADS)

    Weddell, Thomas; Legan, P. Kevin; Lukashkina, Victoria A.; Goodyear, Richard J.; Welstead, Lindsy; Petit, Chistine; Russell, Ian J.; Lukashkin, Andrei N.; Richardson, Guy P.

    2011-11-01

    We demonstrate that in Otoa-/- mice, in which the inner-ear-specific protein otoancorin is absent, excitation of the outer hair cells and cochlear amplification is normal. This finding is remarkable because the tectorial membrane (TM), although remaining functionally attached to the outer hair cell bundles, is completely detached from the spiral limbus. Therefore, as in ancestral vertebrate auditory organs, where inertia provides the excitatory force to the hair cells, it is the inertia of the TM that must be important for exciting the outer hair cells, setting the sensitivity of their transducer conductance, and determining the precise timing of cochlear amplification.

  11. Mice with a deletion of the major central myelin protein exhibit hypersensitivity to noxious thermal stimuli: involvement of central sensitization.

    PubMed

    Petit, Bérengère; Giraudet, Fabrice; Béchon, Céline; Bardin, Laurent; Avan, Paul; Boespflug-Tanguy, Odile; Bégou, Mélina

    2014-05-01

    Null mutations in the gene encoding the major myelin protein of the central nervous system, proteolipid protein 1 (PLP1), cause an X-linked form of spastic paraplegia (SPG2) associated with axonal degeneration. While motor symptoms are the best known manifestations of this condition, its somatosensory disturbances have been described but poorly characterized. We carried out a longitudinal study in an animal model of SPG2 - mice carrying a deletion of the Plp1 gene (Plp-null mice). Plp-null mice exhibited severe early-onset thermal hyperalgesia, in the absence of thermal allodynia. We first performed an electrophysiological testing which showed an early decrease in peripheral and spinal conduction velocities in Plp null mice. Such as the abnormal sensitive behaviors, this slowing of nerve conduction was observed before the development of myelin abnormalities at the spinal level, from 3months of age, and without major morphological defects in the sciatic nerve. To understand the link between a decrease in nerve velocity and an increased response to thermal stimuli before the appearance of myelin abnormalities, we focused our attention on the dorsal horn of the spinal cord, the site of integration of somatosensory information. Immunohistochemical studies revealed an early-onset activation of astrocytes and microglia that worsened with age, associated later in age with perturbation of the expression of the sensory neuropeptides calcitonin-gene-related peptide and galanin. Taken together, these results represent complementary data supporting the hypothesis that Plp-null mice suffer from ganglionopathy associated with late onset central demyelination but with few peripheral nerve alterations, induced by the glial-cell-mediated sensitization of the spinal cord. The mechanism suggested here could underlie pain experiments in other leukodystrophies as well as in other non-genetic demyelinating diseases such as multiple sclerosis. PMID:24423646

  12. Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance.

    PubMed

    Aebischer, T; Morris, L; Handman, E

    1994-10-01

    It is well established that BALB/c mice can be protected from fatal infection with Leishmania major by prophylactic intravenous (i.v.) immunization with irradiated parasites. Protection is critically dependent on the route of injection with i.v. injection being protective and subcutaneous injection not protective. We used this BALB/c-L. major model system to investigate this phenomenon. We analyzed quantitatively the parasite-specific, CD4+ T cell mediated immune responses by limiting dilution. Subcutaneous vaccination resulted in priming of CD4+ precursor T cells, whereas i.v. vaccination was ineffectual. Moreover, i.v. injection prevented the increase in the number of specific precursor cells induced by infection of normal mice during the first weeks post-challenge with virulent parasites. We show here that this was not due to the elimination of the virulent challenge parasites as a result of immunity nor to inefficient antigen presentation of the irradiated organisms after i.v. injection. The data presented here suggest that i.v. injection results in tolerization rather than immunization. Tolerization as a mechanism of host protection is consistent with earlier observations that transient immunosuppression results in cure of L. major infection in BALB/c mice. Transfer of antigen presenting cells (APC) isolated from spleens of mice injected previously with irradiated parasites mimicked to some extent the effect of i.v. immunization with irradiated parasites. The possible involvement of these APC in decreasing the parasite-specific T cell response is discussed. PMID:7826944

  13. 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.

  14. The Multiple Forms of Leishmania major in BALB/C Mice Lung in Iran

    PubMed Central

    Shirbazou, SH; Jafari, M

    2012-01-01

    Cutaneous leishmaniasis is one of the most important parasitic diseases, which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days, the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected. PMID:23109953

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  19. Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

    PubMed

    Lopez, M J; Upchurch, B H; Rindi, G; Leiter, A B

    1995-01-13

    We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells. PMID:7822327

  20. Functions of glycans revealed by gene inactivation of L-selectin ligand sulfotransferases in mice.

    PubMed

    Kawashima, Hiroto

    2006-01-01

    Lymphocyte homing is mediated by a specific interaction between L-selectin and its sulfated glycoprotein ligands expressed on high endothelial venules (HEVs) in lymph nodes. To examine the significance of sulfation of L-selectin ligands, gene targeting mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 (HEC-GlcNAc6ST/ LSST) have been generated. In the double-knockout mice, binding of MECA-79 antibody to lymph node HEV was completely abolished, indicating that extended core 1 O-glycans containing GlcNAc-6-O-sulfate is completely diminished in those mice. Furthermore, the mutant mice showed approximately 75% less lymphocyte homing to the peripheral lymph nodes (PLNs) and significantly less contact hypersensitivity response than wild-type mice, demonstrating that GlcNAc6ST-1 and GlcNAc6ST-2 play a major role in L-selectin ligand biosynthesis in HEVs. In this chapter, the detailed protocols that have been used for the functional assays of these sulfotransferase double-knockout mice are described. PMID:17113873

  1. 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.

  2. Batf3-dependent CD103+ dendritic cells are major producers of IL-12 that drive local Th1 immunity against Leishmania major infection in mice

    PubMed Central

    Martínez-López, María; Iborra, Salvador; Conde-Garrosa, Ruth; Sancho, David

    2015-01-01

    The role of different DC subsets in priming and maintenance of immunity against Leishmania major (L. major) infection is debated. The transcription factor basic leucine zipper transcription factor, ATF-like 3 (Batf3) is essential for the development of mouse CD103+ DCs and some functions of CD8α+ DCs. We found that CD103+ DCs were significantly reduced in the dermis of Batf3-deficient C57BL/6 mice. Batf3−/− mice developed exacerbated and unresolved cutaneous pathology following a low dose of intradermal L. major infection in the ear pinnae. Parasite load was increased 1000-fold locally and expanded systemically. Batf3 deficiency did not affect L. major antigen presentation to T cells, which was directly exerted by CD8α− conventional DCs (cDCs) in the skin draining LN. However, CD4+ T-cell differentiation in the LN and skin was skewed to nonprotective Treg- and Th2-cell subtypes. CD103+ DCs are major IL-12 producers during L. major infection. Local Th1 immunity was severely hindered, correlating with impaired IL-12 production and reduction in CD103+ DC numbers. Adoptive transfer of WT but not IL-12p40−/− Batf3-dependent DCs significantly improved anti-L. major response in infected Batf3−/− mice. Our results suggest that IL-12 production by Batf3-dependent CD103+ DCs is crucial for maintenance of local Th1 immunity against L. major infection. PMID:25312824

  3. Delphinidin, One of the Major Anthocyanidins, Prevents Bone Loss through the Inhibition of Excessive Osteoclastogenesis in Osteoporosis Model Mice

    PubMed Central

    Muramatsu, Masashi; Nomura, Atsushi; Inoue, Fumihide; Into, Takeshi; Yoshiko, Yuji; Niida, Shumpei

    2014-01-01

    Anthocyanins, one of the flavonoid subtypes, are a large family of water-soluble phytopigments and have a wide range of health-promoting benefits. Recently, an anthocyanin-rich compound from blueberries was reported to possess protective property against bone loss in ovariectomized (OVX) animal models. However, the active ingredients in the anthocyanin compound have not been identified. Here we show that delphinidin, one of the major anthocyanidins in berries, is a potent active ingredient in anti-osteoporotic bone resorption through the suppression of osteoclast formation. In vitro examinations revealed that delphinidin treatment markedly inhibited the differentiation of RAW264.7 cells into osteoclasts compared with other anthocyanidins, cyanidin and peonidin. Oral administration of delphinidin significantly prevented bone loss in both RANKL-induced osteoporosis model mice and OVX model mice. We further provide evidence that delphinidin suppressed the activity of NF-κB, c-fos, and Nfatc1, master transcriptional factors for osteoclastogenesis. These results strongly suggest that delphinidin is the most potent inhibitor of osteoclast differentiation and will be an effective agent for preventing bone loss in postmenopausal osteoporosis. PMID:24824988

  4. 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

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

    PubMed

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

    2016-05-01

    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

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

    PubMed Central

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-01-01

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2caflox/flox transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2caflox/flox; Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2caflox/flox; Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2caflox/flox mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases. PMID:27213341

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

    PubMed

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-01-01

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2ca(flox/flox) transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2ca(flox/flox); Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2ca(flox/flox); Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2ca(flox/flox) mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases. PMID:27213341

  8. 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

  9. Dual role of preputial gland secretion and its major components in sex recognition of mice.

    PubMed

    Zhang, Jian-Xu; Liu, Ying-Juan; Zhang, Jin-Hua; Sun, Lixing

    2008-10-20

    This study was aimed at validating the sexual attractiveness of hexadecanol and hexadecyl acetate, two putative pheromone compounds, from preputial gland secretion of mice. These two compounds have been reported to be among the major components of preputial gland secretion in both sexes but higher in quantity in males than females. In this study, we show that castration suppressed the production of the two compounds, further suggesting their association with maleness. Adding preputial gland secretion and the synthetic analogs of the two compounds to castrated male urine at their physiological levels in intact males increased the attractiveness of castrated male urine to female mice, showing that the two compounds were indeed male pheromones. Furthermore, their sexual attractiveness disappeared upon removing the vomeronasal organs (VNOs) from female recipients. Replenishing castrated male urine with preputial gland secretion and the two compounds at their physiological levels in females increased the attractiveness of castrated male urine to males. Such a reversal of sexual attractiveness for hexadecanol and hexadecyl acetate suggests that they had opposing dual effects in sexual attractiveness in a dosage-dependent manner. PMID:18657559

  10. Major Effects on Teratogen-Induced Facial Clefting in Mice Determined by a Single Genetic Region

    PubMed Central

    Karolyi, J.; Erickson, R. P.; Liu, S.; Killewald, L.

    1990-01-01

    A major correlation has been found between the incidence of glucocorticoid-induced cleft palate and the chromosome 8 segment identified by N-acetyl transferase in mice. The resistant strain became fully susceptible while the susceptible strain became resistant when this chromosomal region, representing <0.7% of the genome, was transferred from one strain to the other by the construction of congenic strains. 6-Aminonicotinamide-induced cleft palate and phenytoin-induced cleft lip with or without cleft palate are also influenced by this genetic region but not as strongly. In both cases the susceptible strain became quite resistant to the teratogen-induced clefting when the N-acetyl transferase region of chromosome 8 was transferred. However, this chromosomal region does not make the resistant strain susceptible to these two teratogens. PMID:2227380

  11. 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

  12. αβγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction

    PubMed Central

    Greten-Harrison, Becket; Polydoro, Manuela; Morimoto-Tomita, Megumi; Diao, Ling; Williams, Andrew M.; Nie, Esther H.; Makani, Sachin; Tian, Ning; Castillo, Pablo E.; Buchman, Vladimir L.; Chandra, Sreeganga S.

    2010-01-01

    Synucleins are a vertebrate-specific family of abundant neuronal proteins. They comprise three closely related members, α-, β-, and γ-synuclein. α-Synuclein has been the focus of intense attention since mutations in it were identified as a cause for familial Parkinson's disease. Despite their disease relevance, the normal physiological function of synucleins has remained elusive. To address this, we generated and characterized αβγ-synuclein knockout mice, which lack all members of this protein family. Deletion of synucleins causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. Abrogation of synuclein expression decreased excitatory synapse size by ∼30% both in vivo and in vitro, revealing that synucleins are important determinants of presynaptic terminal size. Young synuclein null mice show improved basic transmission, whereas older mice show a pronounced decrement. The late onset phenotypes in synuclein null mice were not due to a loss of synapses or neurons but rather reflect specific changes in synaptic protein composition and axonal structure. Our results demonstrate that synucleins contribute importantly to the long-term operation of the nervous system and that alterations in their physiological function could contribute to the development of Parkinson's disease. PMID:20974939

  13. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  14. Protective Response to Leishmania major in BALB/c Mice Requires Antigen Processing in the Absence of DM1

    PubMed Central

    Kamala, Tirumalai; Nanda, Navreet K.

    2009-01-01

    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-γ. 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. PMID:19342667

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25452429

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

    PubMed

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

    2016-08-01

    Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes. PMID:26940723

  18. Effect of root bark extract of Berberis vulgaris L. on Leishmania major on BALB/c mice.

    PubMed

    Salehabadi, Alireza; Karamian, Mahdi; Farzad, Motevalli Haghi; Namaei, Mohammad Hasan

    2014-03-01

    Leishmaniasis is one of the most important diseases transmitted by arthropod. This disease is present in 88 countries. Approximately 400 million people are at risk, and 12 million are involved. We aimed to examine the application of ethanolic extract of the root bark of Berberis vulgaris L. for treatment of mice infected with cutaneous leishmaniasis. At first, 40 BALB/c mice were infected to Leishmania major promastigotes and were divided in two groups A and B. Then, each of A and B groups were divided to two subgroups. Mice from subgroup A1 were treated with 10% root bark alcoholic extract, and mice from subgroup A2 were treated with only alcohol (control). Mice from subgroup B1 were treated with 20% root bark alcoholic extract, and mice from subgroup B2 were treated with only alcohol (control). The 90% recovery was found in the mice treated with 20% root bark extract, and 55% recovery was found with 10% root bark extract, but in the control group, 0% recovery was found. The results of our study showed that the lotion of root bark extract has good suppression effects on parasites. Therefore, it might be a pro for developing new antileishmanial drugs. PMID:24337510

  19. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice*

    PubMed Central

    Bartoi, Tudor; Rigbolt, Kristoffer T. G.; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-01-01

    GABAB receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABAB1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABAB receptors. The transgenic mice express GABAB1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABAB1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABAB receptors via the GABAB2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABAB2. The mice equipped with tags on GABAB1 facilitate validation and identification of native binding partners of GABAB receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  20. GABAB receptor constituents revealed by tandem affinity purification from transgenic mice.

    PubMed

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-07-01

    GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  1. 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

  2. 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

  3. 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

  4. Neuroinformatics analyses reveal GABAt and SSADH as major proteins involved in anticonvulsant activity of valproic acid.

    PubMed

    Piplani, Sakshi; Verma, Prabhakar Kumar; Kumar, Ajit

    2016-07-01

    The unequivocal hypotheses about anticonvulsant activity of valproic acid (VPA) have always been a basic hurdle in designing next generation neurotherapeutics, particularly the anti-epileptic drugs. The present study reports about a comprehensive in-silico investigation into qualitative and quantitative binding of VPA and corresponding natural ligands of four major enzymes involved in neurotransmissions, namely-GABA transaminase (GABAt), α-keto glutarate dehydrogenase (α-KGDH), Succinate Semialdehyde dehydrogenase (SSADH) and Glutamate Decarboxylase (GAD), respectively. The molecular docking analyses revealed that VPA inhibits GABAt and α-KGDH through allosteric while SSADH through competitive mode of binding. There is an observed elevation in binding of glutamate over GAD in the presence of VPA. The docking inhibition constant (Ki) of VPA to all the studied enzymatic receptors were observed to be well below the therapeutic concentration of VPA in blood, except for α-KGDH, thus favouring GABAergic over glutamatergic mode of anticonvulsant activity of VPA. The report is probably the first comprehensive in-silico molecular study about VPA action. PMID:27261619

  5. 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.

  6. 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.

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

    PubMed

    Masic, Anita; Valencia Hernandez, Ana Maria; 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

  8. 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

  9. 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. PMID:26869186

  10. Major Basic Protein from Eosinophils and Myeloperoxidase from Neutrophils Are Required for Protective Immunity to Strongyloides stercoralis in Mice

    PubMed Central

    O'Connell, Amy E.; Hess, Jessica A.; Santiago, Gilberto A.; Nolan, Thomas J.; Lok, James B.; Lee, James J.; Abraham, David

    2011-01-01

    Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity. PMID:21482685

  11. 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.

  12. 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

  13. Cerebellar Transcriptome Profiles of ATXN1 Transgenic Mice Reveal SCA1 Disease Progression and Protection Pathways.

    PubMed

    Ingram, Melissa; Wozniak, Emily A L; Duvick, Lisa; Yang, Rendong; Bergmann, Paul; Carson, Robert; O'Callaghan, Brennon; Zoghbi, Huda Y; Henzler, Christine; Orr, Harry T

    2016-03-16

    SCA1, a fatal neurodegenerative disorder, is caused by a CAG expansion encoding a polyglutamine stretch in the protein ATXN1. We used RNA sequencing to profile cerebellar gene expression in Pcp2-ATXN1[82Q] mice with ataxia and progressive pathology and Pcp2-ATXN1[30Q]D776 animals having ataxia in absence of Purkinje cell progressive pathology. Weighted Gene Coexpression Network Analysis of the cerebellar expression data revealed two gene networks that significantly correlated with disease and have an expression profile correlating with disease progression in ATXN1[82Q] Purkinje cells. The Magenta Module provides a signature of suppressed transcriptional programs reflecting disease progression in Purkinje cells, while the Lt Yellow Module reflects transcriptional programs activated in response to disease in Purkinje cells as well as other cerebellar cell types. Furthermore, we found that upregulation of cholecystokinin (Cck) and subsequent interaction with the Cck1 receptor likely underlies the lack of progressive Purkinje cell pathology in Pcp2-ATXN1[30Q]D776 mice. PMID:26948890

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

    SciTech Connect

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

    1985-01-01

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

  15. Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen.

    PubMed

    Pichurin, Pavel N; Pichurina, Oxana; Marians, Russell C; Chen, Chun-Rong; Davies, Terry F; Rapoport, Basil; McLachlan, Sandra M

    2004-03-01

    Graves' disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-gamma production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance. PMID:14630711

  16. 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. PMID:26542948

  17. 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

    2015-07-01

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

  18. Influence of 5 major Salmonella pathogenicity islands on NK cell depletion in mice infected with Salmonella enterica serovar Enteritidis

    PubMed Central

    2010-01-01

    Background In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants. Results The virulence of S. Enteritidis for Balb/C mice was exclusively dependent on intact SPI-2. Infections with any of the mutants harbouring SPI-2 (including the mutant in which we left only SPI-2 but removed SPI-1, SPI-3, SPI-4 and SPI-5) resulted in fatalities, liver injures and NK cell depletion from the spleen. The infection was of minimal influence on counts of splenic CD4 CD8 T lymphocytes and γδ T-lymphocytes although a reduced ability of splenic lymphocytes to respond to non-specific mitogens indicated general immunosuppression in mice infected with SPI-2 positive S. Enteritidis mutants. Further investigations showed that NK cells were depleted also in blood but not in the caecal lamina propria. However, NK cell depletion was not directly associated with the presence of SPI-2 and was rather an indicator of virulence or avirulence of a particular mutant because the depletion was not observed in mice infected with other attenuated mutants such as lon and rfaL. Conclusions The virulence of S. Enteritidis for Balb/C mice is exclusively dependent on the presence of SPI-2 in its genome, and a major hallmark of the infection in terms of early changes in lymphocyte populations is the depletion of NK cells in spleen and blood. The decrease of NK cells in circulation can be used as a marker of attenuation of S. Enteritidis mutants for Balb/C mice. PMID:20226037

  19. Temporal fate mapping reveals age-linked heterogeneity in naive T lymphocytes in mice

    PubMed Central

    Hogan, Thea; Gossel, Graeme; Yates, Andrew J.; Seddon, Benedict

    2015-01-01

    Understanding how our T-cell compartments are maintained requires knowledge of their population dynamics, which are typically quantified over days to weeks using the administration of labels incorporated into the DNA of dividing cells. These studies present snapshots of homeostatic dynamics and have suggested that lymphocyte populations are heterogeneous with respect to rates of division and/or death, although resolving the details of such heterogeneity is problematic. Here we present a method of studying the population dynamics of T cells in mice over timescales of months to years that reveals heterogeneity in rates of division and death with respect to the age of the host at the time of thymic export. We use the transplant conditioning drug busulfan to ablate hematopoetic stem cells in young mice but leave the peripheral lymphocyte compartments intact. Following their reconstitution with congenically labeled (donor) bone marrow, we followed the dilution of peripheral host T cells by donor-derived lymphocytes for a year after treatment. Describing these kinetics with mathematical models, we estimate rates of thymic production, division and death of naive CD4 and CD8 T cells. Population-averaged estimates of mean lifetimes are consistent with earlier studies, but we find the strongest support for a model in which both naive T-cell pools contain kinetically distinct subpopulations of older host-derived cells with self-renewing capacity that are resistant to displacement by naive donor lymphocytes. We speculate that these incumbent cells are conditioned or selected for increased fitness through homeostatic expansion into the lymphopenic neonatal environment. PMID:26607449

  20. Temporal fate mapping reveals age-linked heterogeneity in naive T lymphocytes in mice.

    PubMed

    Hogan, Thea; Gossel, Graeme; Yates, Andrew J; Seddon, Benedict

    2015-12-15

    Understanding how our T-cell compartments are maintained requires knowledge of their population dynamics, which are typically quantified over days to weeks using the administration of labels incorporated into the DNA of dividing cells. These studies present snapshots of homeostatic dynamics and have suggested that lymphocyte populations are heterogeneous with respect to rates of division and/or death, although resolving the details of such heterogeneity is problematic. Here we present a method of studying the population dynamics of T cells in mice over timescales of months to years that reveals heterogeneity in rates of division and death with respect to the age of the host at the time of thymic export. We use the transplant conditioning drug busulfan to ablate hematopoetic stem cells in young mice but leave the peripheral lymphocyte compartments intact. Following their reconstitution with congenically labeled (donor) bone marrow, we followed the dilution of peripheral host T cells by donor-derived lymphocytes for a year after treatment. Describing these kinetics with mathematical models, we estimate rates of thymic production, division and death of naive CD4 and CD8 T cells. Population-averaged estimates of mean lifetimes are consistent with earlier studies, but we find the strongest support for a model in which both naive T-cell pools contain kinetically distinct subpopulations of older host-derived cells with self-renewing capacity that are resistant to displacement by naive donor lymphocytes. We speculate that these incumbent cells are conditioned or selected for increased fitness through homeostatic expansion into the lymphopenic neonatal environment. PMID:26607449

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-29

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

  3. Unsuspected early neuronal loss in scrapie-infected mice revealed by morphometric analysis.

    PubMed

    Scott, J R; Jeffrey, M; Halliday, W G

    1994-06-01

    This study was undertaken to determine to what extent neuronal loss is a feature of scrapie pathology, using an experimental model in which infectivity and subsequent vacuolar lesions are well characterized but in which neuronal loss has not been previously identified. Intraocular infection with ME7 scrapie directs infection through the major projections of the optic nerve, which include the dorsal lateral geniculate nucleus (dLGN) on the contralateral side to the infected eye. Infectivity can be detected in the dLGN at 77 days post-infection and vacuolar lesions are first seen around halfway through the incubation period of 240 days. Morphometric assessment of neuron number in the dLGN was made on gallocyanin stained semi-serial sections from 5 infected and 5 normal brain-injected controls at 4 fifty-day intervals during the incubation period, and on clinically terminal mice. The number of neurons in the dLGN of the infected mice decreased steadily from around 20,000 at 50 days post-infection to under 2,000 in the terminal group. The loss was delayed in the ipsilateral dLGN, although terminal counts were the same for both sides. The onset of neuronal loss was coincident with initial vacuolar changes, and neuronal numbers were inversely proportional to the severity of vacuolation. It is concluded that scrapie infection causes a progressive neuronal loss that can be identified some 30-80 days after infectivity can be detected in the dLGN, long before the onset of clinical disease. PMID:8030955

  4. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis.

    PubMed

    Mill, Jonathan; Tang, Thomas; Kaminsky, Zachary; Khare, Tarang; Yazdanpanah, Simin; Bouchard, Luigi; Jia, Peixin; Assadzadeh, Abbas; Flanagan, James; Schumacher, Axel; Wang, Sun-Chong; Petronis, Arturas

    2008-03-01

    Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder. PMID:18319075

  5. Increased cortical-limbic anatomical network connectivity in major depression revealed by diffusion tensor imaging.

    PubMed

    Fang, Peng; Zeng, Ling-Li; Shen, Hui; Wang, Lubin; Li, Baojuan; Liu, Li; Hu, Dewen

    2012-01-01

    Magnetic resonance imaging studies have reported significant functional and structural differences between depressed patients and controls. Little attention has been given, however, to the abnormalities in anatomical connectivity in depressed patients. In the present study, we aim to investigate the alterations in connectivity of whole-brain anatomical networks in those suffering from major depression by using machine learning approaches. Brain anatomical networks were extracted from diffusion magnetic resonance images obtained from both 22 first-episode, treatment-naive adults with major depressive disorder and 26 matched healthy controls. Using machine learning approaches, we differentiated depressed patients from healthy controls based on their whole-brain anatomical connectivity patterns and identified the most discriminating features that represent between-group differences. Classification results showed that 91.7% (patients=86.4%, controls=96.2%; permutation test, p<0.0001) of subjects were correctly classified via leave-one-out cross-validation. Moreover, the strengths of all the most discriminating connections were increased in depressed patients relative to the controls, and these connections were primarily located within the cortical-limbic network, especially the frontal-limbic network. These results not only provide initial steps toward the development of neurobiological diagnostic markers for major depressive disorder, but also suggest that abnormal cortical-limbic anatomical networks may contribute to the anatomical basis of emotional dysregulation and cognitive impairments associated with this disease. PMID:23049910

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

    PubMed

    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

  7. 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

  8. Whole brain resting-state analysis reveals decreased functional connectivity in major depression.

    PubMed

    Veer, Ilya M; Beckmann, Christian F; van Tol, Marie-José; Ferrarini, Luca; Milles, Julien; Veltman, Dick J; Aleman, André; van Buchem, Mark A; van der Wee, Nic J; Rombouts, Serge A R B

    2010-01-01

    Recently, both increases and decreases in resting-state functional connectivity have been found in major depression. However, these studies only assessed functional connectivity within a specific network or between a few regions of interest, while comorbidity and use of medication was not always controlled for. Therefore, the aim of the current study was to investigate whole-brain functional connectivity, unbiased by a priori definition of regions or networks of interest, in medication-free depressive patients without comorbidity. We analyzed resting-state fMRI data of 19 medication-free patients with a recent diagnosis of major depression (within 6 months before inclusion) and no comorbidity, and 19 age- and gender-matched controls. Independent component analysis was employed on the concatenated data sets of all participants. Thirteen functionally relevant networks were identified, describing the entire study sample. Next, individual representations of the networks were created using a dual regression method. Statistical inference was subsequently done on these spatial maps using voxel-wise permutation tests. Abnormal functional connectivity was found within three resting-state networks in depression: (1) decreased bilateral amygdala and left anterior insula connectivity in an affective network, (2) reduced connectivity of the left frontal pole in a network associated with attention and working memory, and (3) decreased bilateral lingual gyrus connectivity within ventromedial visual regions. None of these effects were associated with symptom severity or gray matter density. We found abnormal resting-state functional connectivity not previously associated with major depression, which might relate to abnormal affect regulation and mild cognitive deficits, both associated with the symptomatology of the disorder. PMID:20941370

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

    PubMed

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

    2016-01-22

    Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions. PMID:26602167

  10. Dual elimination of the glucagon and GLP-1 receptors in mice reveals plasticity in the incretin axis.

    PubMed

    Ali, Safina; Lamont, Benjamin J; Charron, Maureen J; Drucker, Daniel J

    2011-05-01

    Disordered glucagon secretion contributes to the symptoms of diabetes, and reduced glucagon action is known to improve glucose homeostasis. In mice, genetic deletion of the glucagon receptor (Gcgr) results in increased levels of the insulinotropic hormone glucagon-like peptide 1 (GLP-1), which may contribute to the alterations in glucose homeostasis observed in Gcgr-/- mice. Here, we assessed the contribution of GLP-1 receptor (GLP-1R) signaling to the phenotype of Gcgr-/- mice by generating Gcgr-/-Glp1r-/- mice. Although insulin sensitivity was similar in all genotypes, fasting glucose was increased in Gcgr-/-Glp1r-/- mice. Elimination of the Glp1r normalized gastric emptying and impaired intraperitoneal glucose tolerance in Gcgr-/- mice. Unexpectedly, deletion of Glp1r in Gcgr-/- mice did not alter the improved oral glucose tolerance and increased insulin secretion characteristic of that genotype. Although Gcgr-/-Glp1r-/- islets exhibited increased sensitivity to the incretin glucose-dependent insulinotropic polypeptide (GIP), mice lacking both Glp1r and the GIP receptor (Gipr) maintained preservation of the enteroinsular axis following reduction of Gcgr signaling. Moreover, Gcgr-/-Glp1r-/- islets expressed increased levels of the cholecystokinin A receptor (Cckar) and G protein-coupled receptor 119 (Gpr119) mRNA transcripts, and Gcgr-/-Glp1r-/- mice exhibited increased sensitivity to exogenous CCK and the GPR119 agonist AR231453. Our data reveal extensive functional plasticity in the enteroinsular axis via induction of compensatory mechanisms that control nutrient-dependent regulation of insulin secretion. PMID:21540554

  11. 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

  12. 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. PMID:27120982

  13. Inoculation of killed Leishmania major into immune mice rapidly disrupts immunity to a secondary challenge via IL-10-mediated process

    PubMed Central

    Okwor, Ifeoma; Liu, Dong; Beverley, Stephen M.; Uzonna, Jude E.

    2009-01-01

    Recovery from natural or experimental Leishmania major infection, the causative agent of cutaneous leishmaniasis, results in development of durable immunity in mice and humans that is manifested as rapid control of parasite replication and resolution of cutaneous lesion after secondary challenge. This form of “infection-induced” immunity is thought to occur naturally in endemic areas and is generally considered the gold standard for any effective vaccine against cutaneous leishmaniasis. To determine factors that might heighten or abrogate infection-induced immunity, we investigated the impact of inoculating dead antigen in the form of killed Leishmania parasites to healed mice. We show that inoculation of killed parasites into mice that resolved their primary virulent L. major infection results in rapid and relatively sustained loss of infection-induced immunity. This loss of immunity was not due to the inability of killed parasites to induce inflammatory responses (such as delayed type hypersensitivity), but it was related to their failure to induce robust IFN-γ response. Furthermore, inoculation of killed Leishmania parasites into healed mice led to rapid expansion of IL-10-producing CD4+CD25+Foxp3+ T cells in lymph nodes draining the primary infection site. Treatment with anti-CD25 or anti-IL-10R mAb abolished killed parasite-induced loss of immunity. Our study suggests that vaccination with killed parasites could predispose naturally immune individuals to become susceptible to new infections and/or disease reactivation. This may account for the lack of efficacy of such vaccines in field trials in endemic regions. These findings have important implications for vaccine design and vaccination strategies against human cutaneous leishmaniasis. PMID:19666482

  14. 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

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

    PubMed Central

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

    2014-01-01

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

  16. Whole-genome expression analysis reveals genes associated with treatment response to escitalopram in major depression.

    PubMed

    Pettai, Kristi; Milani, Lili; Tammiste, Anu; Võsa, Urmo; Kolde, Raivo; Eller, Triin; Nutt, David; Metspalu, Andres; Maron, Eduard

    2016-09-01

    The reasons for variability in treatment response in major depressive disorder (MDD) are not fully understood, but there is accumulating evidence suggesting that therapeutic outcomes of antidepressants can be influenced by genetic factors. In the present study we applied the microarray Illumina platform for whole genome expression profiling in depressive patients treated with escitalopram medication in order to identify genes underlying response to antidepressant treatment. The initial study sample consisted of 135 outpatients with major depressive disorder (mean age 31.1±11.6 years, 68% females) treated with escitalopram 10-20mg/day for 12 weeks, from which 87 patients (55 females) were included in gene expression analyzing. The gene expression profiles were measured on peripheral blood cells at baseline, at week 4 and at the end of treatment (week 12) using BeadChips Illumina. The fold change was used to demonstrate rate of changes in average gene expressions between studied groups. Statistical analyses were performed using the false discovery rate (FDR). The most interesting gene, which showed the predictive effect on treatment outcome by delineating low dose responders and treatment-resistant patients at the beginning of medication, was NLGN2, belonging to a family of neuronal cell surface proteins and involving in synapse formation. In addition, the several gene clusters, related to immune response, signal transduction and neurotrophin pathway, have distinguished responders from non-responders at the week 4 of treatment. After 4 weeks of escitalopram treatment (10mg/day), the YWHAZ gene has showed the highest transcriptional change in responders as compared with non-responders. Finally, at the end of the treatment we noticed that at least three genes (NR2C2, ZNF641, FKBP1A) have been strongly associated with resistance to escitalopram. Thus the results of this study support that exploration of peripheral gene expression is a useful tool in the further

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

    PubMed

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

    2014-03-10

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

  18. Proteomics reveals major components of oogenesis in the reproductive tract of sugar-fed Anopheles aquasalis.

    PubMed

    Dias-Lopes, Geovane; Borges-Veloso, Andre; Saboia-Vahia, Leonardo; Padrón, Gabriel; de Faria Castro, Cássia Luana; Guimarães, Ana Carolina Ramos; Britto, Constança; Cuervo, Patricia; De Jesus, Jose Batista

    2016-05-01

    Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main tool for interrupting parasite transmission. Mechanisms of oogenesis and embryogenesis in anautogenous mosquitoes are mainly modulated by blood feeding. However, the expression, at the protein level, of genes involved in such mechanisms in sugar-fed females is unknown. In this work, total protein extracts of the reproductive tract of female An. aquasalis that were fed sugar were analyzed using liquid chromatography followed by mass spectrometry for protein identification and bioinformatic tools for data mining. We identified 922 proteins expressed in the organ, and using several databases, we attributed biological meaning for several of them. Remarkably, nine proteins involved in oogenesis were identified in females fed sugar. Putative vitellogenins, vitellogenin receptor, lipid storage droplet, transferrin, ferritin, and apolipoprotein, identified here, are proteins involved in egg development. Proteins involved in embryonic development, such as paxillin, exuperantia, several growth factors, and dorsal switch protein, were identified. Interestingly, in this study, we identified 15 peptidases of various classes such as aminopeptidases, carboxypeptidases, serine protease, cathepsin, and metalloprotease that could potentially interact with male seminal components. Here, we demonstrated that the reproductive tract of female An. aquasalis fed on sugar expresses proteins involved in oogenesis and embryonic development. These findings reveal unknown aspects of the physiology of this organ under the given nutritional conditions. PMID:26850722

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

    PubMed Central

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

    2015-01-01

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

  20. FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major.

    PubMed

    Sterkers, Yvon; Lachaud, Laurence; Crobu, Lucien; Bastien, Patrick; Pagès, Michel

    2011-02-01

    The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity. PMID:20964798

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

    PubMed

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

    2015-04-01

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

  2. Electrical brain imaging reveals the expression and timing of altered error monitoring functions in major depression.

    PubMed

    Aarts, Kristien; Vanderhasselt, Marie-Anne; Otte, Georges; Baeken, Chris; Pourtois, Gilles

    2013-11-01

    Major depressive disorder (MDD) is characterized by disturbances in affect, motivation, and cognitive control processes, including error detection. However, the expression and timing of the impairments during error monitoring remain unclear in MDD. The behavior and event-related brain responses (ERPs) of 20 patients with MDD were compared with those of 20 healthy controls (HCs), while they performed a Go/noGo task. Errors during this task were associated with 2 ERP components, the error-related negativity (ERN/Ne) and the error positivity (Pe). Results show that the ERN/Ne-correct-related negativity (CRN) amplitude difference was significantly larger in MDD patients (after controlling for speed), compared with HCs, although MDD patients exhibited overactive medial frontal cortex (MFC) activation. By comparison, the subsequent Pe component was smaller in MDD patients compared with HCs and this effect was accompanied by a reduced activation of ventral anterior cingulate cortex (ACC) regions. These results suggest that MDD has multiple cascade effects on early error monitoring brain mechanisms. PMID:24364597

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

    PubMed Central

    2013-01-01

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

  4. 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. PMID:22725256

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

    PubMed

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

    2015-01-01

    Rodents show a stronger preference for fat than sucrose, even if their diet is isocaloric. This implies that the preference mechanisms for fat and sucrose differ. To compare the contribution of the opioid system to the preference of fat and sucrose, we examined the effects of mu-, delta-, kappa-, and non-selective opioid receptor antagonists on the preference of sucrose and fat, assessed by a two-bottle choice test and a licking test, in mice naïve to sucrose and fat ingestion. Administration of non-selective and mu-selective opioid receptor antagonists more strongly inhibited the preference of fat than sucrose. While the preference of fat was reduced to the same level as water by the antagonist administration that of sucrose was still greater than water. Our results suggest that the preference of fat relies strongly on the opioid system, while that of sucrose is regulated by other mechanisms in addition to the opioid system. PMID:25516200

  6. Arsenic trioxide attenuated the rejection of major histocompatibility complex fully-mismatched cardiac allografts in mice.

    PubMed

    Yan, S; Zhang, Q Y; Zhou, B; Xue, L; Chen, H; Wang, Y; Zheng, S S

    2009-06-01

    We investigated the effects of arsenic trioxide (As(2)O(3)) on allogeneic immune response using a mouse heart transplantation model. Mice were randomly divided into 4 groups of 6 animals each. The control group received phosphate-buffered saline (PBS); the As(2)O(3)-treated group, intraperitoneal (IP) injection of As(2)O(3) (1 mg/kg) from days -3 to 10 after heart transplantation. The cyclosporine (CsA)-treated group was given a subtherapeutic dose of CsA (10 mg/kg) IP, and the As(2)O(3) plus CsA-treated group, a combined protocol of As(2)O(3) and CsA. Six days after transplantation, cardiac allografts were harvested for immunohistology and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The survival of the allografts was significantly improved among the As(2)O(3)-treated group compared with the control group (17.2 +/- 1.9 vs 8.0 +/- 0.9 days; P < .05). A marked prolongation (28.6 +/- 6.0 days) of graft survival was achieved by the combined protocol compared with the CsA-treated group (9.6 +/- 3.0 days; P < .05) or the As(2)O(3)-treated group. Allografts of As(2)O(3)-treated and As(2)O(3) plus CsA-treated mice showed a changing pattern of Th1/Th2 cytokine mRNA expression. Allograft rejection was apparently alleviated by low-dose As(2)O(3), and particularly when combined with a subtherapeutic CsA dose. PMID:19545743

  7. 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

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

    PubMed Central

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

    1996-01-01

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

  9. Major Subglacial Meltwater Channels Reveal Former Dynamic Ice Sheet in West Antarctica

    NASA Astrophysics Data System (ADS)

    Rose, Kathryn; Ross, Neil; Bingham, Robert; Corr, Hugh; Ferraccioli, Fausto; Jordan, Tom; LeBrocq, Anne; Rippin, David; Siegert, Martin

    2014-05-01

    The Eocene-Oligocene boundary (ca. 34 Ma) marks the onset of widespread, continental-scale glaciation in Antarctica, due to declining atmospheric carbon dioxide levels and the opening of the Drake Passage. The marine-based West Antarctic Ice Sheet (WAIS) is considered highly susceptible to change, experiencing numerous oscillations since its formation. In order to assess how past changes to the WAIS are relevant for understanding its future behaviour, it is important to comprehend the glaciological processes involved in those changes. Central to this is an appreciation of climate and ice flow regimes, in particular the extent to which former ice sheets have experienced surface melting (as in Greenland today). Geomorphic analysis of subglacial topography has played a key role in reconstructing the nature of former ice masses in Antarctica, as landscape form can be linked to glacial process. While radio-echo sounding (RES) is the primary tool used to map boundary conditions beneath ice sheets, recent developments have demonstrated that satellite imagery of the ice surface can provide insights into subglacial topography, where RES is unavailable. Using this combination of datasets, we have identified a series of major, elongate subglacial features, which we interpret as preserved subglacial channels, developed through the action of water. They are incised into a subglacial plateau in the region between the Möller and Foundation ice streams (MIS and FIS, respectively), in West Antarctica. The channels are observed across an area of ~17,700 km2 and extend 200 km inland from the grounding line. They are located below sea level and track over present-day reverse slopes, indicating a subglacial (rather than pre-glacial) fluvial origin. In order to form, these channels require significant, probably periodic (seasonal), meltwater inputs to the base of the ice sheet. We suggest the channels are the result of meltwater inputs to the subglacial environment from the ice surface

  10. 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

  11. 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. PMID:26938775

  12. Spontaneous Recovery of Pathogenicity by Leishmania major hsp100−/− Alters the Immune Response in Mice

    PubMed Central

    Reiling, Linda; Jacobs, Thomas; Kroemer, Manfred; Gaworski, Iris; Graefe, Sebastian; Clos, Joachim

    2006-01-01

    By using repeated mouse infection cycles, we obtained an escape variant with restored infectivity and pathogenicity that originated from a single, noninfectious hsp100−/− gene (formerly known as ΔclpB) replacement clone of Leishmania major, the causative agent of cutaneous leishmaniasis. This isolate elicited increased infiltration of immune cells to the site of infection and altered the polarization of the immune response in BALB/c mice from a predominantly TH2 type to a TH1 type. A clonal analysis resulted in isolation of two clones with antagonistic properties. While one clone exhibited restored infectivity in isolated macrophages but caused no persistent infection in the mouse model, the second clone was unable to infect macrophages in vitro but could establish a lasting infection and form progressive lesions in BALB/c mice. Our results add to the evidence that the TH1-TH2 dichotomy of the early immune response against L. major not only depends on the genetic predisposition of the host but also depends on intrinsic properties of the parasite. PMID:17057085

  13. 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. PMID:23467583

  14. The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

    PubMed Central

    Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-01-01

    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. PMID:23467583

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

    PubMed

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

    2015-03-01

    Leishmania major is resistant to the traditional treatments in many parts of the world. PgpA, a member of (ABC) transporter superfamily, has been identified in Leishmania involved in antimony resistance. Silymarin can inhibit PgpA. The aim of this study was to determine the effect of combined therapy with glucantime and silymarin on Cutaneous Leishmaniasis. The effects of silymarin on response of L. major to glucantime were evaluated with amastigote macrophage and mice model of leishmaniasis. Immediately after injection in mice inoculated into footpads with L. major amastigote, systemic treatment was performed and the size of footpad swelling was measured twice a week. 4 and 8 weeks after the beginning of the treatment, splenic parasite burden was done. Silymarin showed no significant effect on the response of L. major promastigotes to glucantime. 2 formulations (glucantime 25 µm with silymarin 25 µm or 12.5 µm) reduced cell death in amastigote assays. The effect of silymarin on footpad swelling was detected when the combination of low-dose glucantime (20 mg/kg) with 25-50 mg/kg silymarin (especially 50 mg/kg) were used at day 22 of post infection (P<0.05). According to the parasite burden data, use of silymarin in the presence of different doses of glucantime, did not show significant effect compared to glucantime alone. The results of this study suggest that silymarin in conjunction with glucantime may have benefit effects in murine model of cutaneous leishmaniasis. PMID:24623031

  16. Imaging Mass Spectrometry Reveals a Decrease of Cardiolipin in the Kidney of NASH Model Mice.

    PubMed

    Hayasaka, Takahiro; Fuda, Hirotoshi; Hui, Shu-Ping; Chiba, Hitoshi

    2016-01-01

    Non-alcoholic steatohepatitis (NASH) can be complicated with chronic kidney disease (CKD). In this study, changes in the distribution of biomolecules in the kidney were studied in NASH model mice with the use of imaging mass spectrometry (IMS). The mass spectra and ion images of IMS showed that the signals of cardiolipin (CL) species were decreased in the kidney cortex of the NASH mice. The decrease of CL might therefore suggest the kidney involvement of NASH. PMID:27063723

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Stout, Michael B; Swindell, William R; Zhi, Xu; Rohde, Kyle; List, Edward O; Berryman, Darlene E; Kopchick, John J; Gesing, Adam; Fang, Yimin; Masternak, Michal M

    2015-09-29

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

  19. Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes.

    PubMed

    Wewer Albrechtsen, Nicolai J; Kuhre, Rune E; Windeløv, Johanne A; Ørgaard, Anne; Deacon, Carolyn F; Kissow, Hannelouise; Hartmann, Bolette; Holst, Jens J

    2016-08-01

    Glucagon is a metabolically important hormone, but many aspects of its physiology remain obscure, because glucagon secretion is difficult to measure in mice and rats due to methodological inadequacies. Here, we introduce and validate a low-volume, enzyme-linked immunosorbent glucagon assay according to current analytical guidelines, including tests of sensitivity, specificity, and accuracy, and compare it, using the Bland-Altman algorithm and size-exclusion chromatography, with three other widely cited assays. After demonstrating adequate performance of the assay, we measured glucagon secretion in response to intravenous glucose and arginine in anesthetized mice (isoflurane) and rats (Hypnorm/midazolam). Glucose caused a long-lasting suppression to very low values (1-2 pmol/l) within 2 min in both species. Arginine stimulated secretion 8- to 10-fold in both species, peaking at 1-2 min and returning to basal levels at 6 min (mice) and 12 min (rats). d-Mannitol (osmotic control) was without effect. Ketamine/xylazine anesthesia in mice strongly attenuated (P < 0.01) α-cell responses. Chromatography of pooled plasma samples confirmed the accuracy of the assay. In conclusion, dynamic analysis of glucagon secretion in rats and mice with the novel accurate sandwich enzyme-linked immunosorbent assay revealed extremely rapid and short-lived responses to arginine and rapid and profound suppression by glucose. PMID:27245336

  20. Alterations in Hepatic Metabolism in fld Mice Reveal a Role for Lipin 1 in Regulating VLDL-Triacylglyceride Secretion

    PubMed Central

    Chen, Zhouji; Gropler, Matthew C.; Norris, Jin; Lawrence, John C.; Harris, Thurl E.; Finck, Brian N.

    2009-01-01

    Objective Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. Methods and Results Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1—deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. Conclusions Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance. PMID:18669885

  1. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed Central

    Murray, P J; Spithill, T W; Handman, E

    1989-01-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens. Images PMID:2731987

  2. 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

  3. Park2-null/tau transgenic mice reveal a functional relationship between parkin and tau.

    PubMed

    Guerrero, Rosa; Navarro, Paloma; Gallego, Eva; Avila, Jesus; de Yebenes, Justo G; Sanchez, Marina P

    2008-03-01

    Mutations, haplotypes, and polymorphisms of tau and Park-2 genes constitute risk factors for developing tauopathies. In order to analyze the possible relationship between parkin and tau we generated a double-mutant mouse deficient for Park-2 expression and overexpressing a mutant tau protein (hTauVLW). Mice develop normally, although the median survival rate is considerably reduced with respect to wild type (45%). Aggregates of phosphorylated tau in neurons and reactive gliosis are quite abundant in cortex and hippocampus of these mice. Moreover, while in young transgenic mice the hTauVLW immunostained transgene product is observed in both cell bodies and dendrites, the hTauVLW mutant protein is only detected in the neuronal cell bodies when Park-2 gene is additionally deleted. Moreover, DNA fragmentation was detected by the TUNEL method, and cerebral atrophy is also present in these regions. The levels of phosphorylated tau and Hsp70 are increased in the double-mutant mice, while CHIP expression in hippocampus is lower when the Park-2 gene is deleted. Thus, the combination of Park-2 gene deletion with hTauVLW transgene overexpression in mice produces serious neuropathological effects, which reflect the existence of some relationship between both proteins. PMID:18376058

  4. 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. PMID:24780938

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

    PubMed

    Takeo, Toru; Nakagata, Naomi

    2016-09-15

    Improvement of the superovulation technique will help to enhance the efficiency of embryo and animal production. Blocking inhibin using inhibin antiserum (IAS) is known to promote follicular development by increasing the level of FSH. Previously, we reported that coadministration of IAS and eCG produced more than 100 oocytes from a single female C57BL/6 mouse at 4 weeks old. The oocytes derived from the IAS + eCG (IASe) treatment were able to fertilize and develop normally into offspring. In this study, we examined the effect of IASe treatment on the numbers of ovulated oocytes in major inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J, and FVB/NJ) and outbred (CD1) mice strains at 4 weeks old. We confirmed the fertilization and developmental ability of the IASe-derived oocytes. IASe treatment ovulated 1.5 to 3.2 times higher numbers of oocytes than eCG treatment alone. The fertilization rate of IASe-derived oocytes was similar to that of eCG-derived oocytes. In vitro and in vivo developmental rates of the embryos derived from IASe were similar to the rates of embryos derived from eCG. We have shown that superovulation by IASe is very effective in obtaining high numbers of ovulated oocytes from small numbers of oocyte donor in a number of mice strains. The superovulation technique will contribute to the archiving of cryopreserved embryos of genetically engineered mice using small numbers of donors and has the potential to produce more live animals for rederivation of the archived mouse lines in mouse repositories. PMID:27242176

  6. Plasma metabolic signatures reveal the regulatory effect of exercise training in db/db mice.

    PubMed

    Xiang, L; Cheang, W S; Lin, S H; Wang, L; Li, Y L; Huang, Y; Cai, Z W

    2015-09-01

    Type 2 diabetes (T2DM) is caused by a complex set of interactions between genetic modifications and life styles. This complexity creates challenges for a full understanding of the altered metabolic pathways that contribute to the development of T2DM, which needs a comprehensive metabolic analysis. Exercise training is a common therapeutic approach known to antagonize the metabolic consequences of T2DM. However, the metabolic phenotypes of exercise effected in T2DM have not been clearly characterized. Here, we present the effect of physical activity on biochemical changes in diabetic db/db mice. An untargeted metabolomics study based on liquid chromatography coupled with high resolution mass spectrometry was carried out to delineate the plasma metabolic signatures in conjunction with a multivariate statistical analysis. As a result, a total of 24 differential metabolites were identified, covering amino acids, organic acids and lipids. Three biomarkers, including lysine, creatine and uridine, were significantly reversed by exercise training in db/db diabetic mice groups compared to lean db/m+ groups. Of note, pantothenic acid and palmitoylcarnitine, which are involved in fatty acid β-oxidation (FAO), were promoted by exercise training in diabetic mice rather than in lean mice. These findings indicated that diabetic mice might be more susceptible to exercise for energy expenditure. Together, the results might demonstrate that exercise could mitigate insulin resistance in T2DM through improving FAO and that uridine in blood might be an important indicator to reflect insulin sensitivity promoted by exercise training in T2DM mice. PMID:26227879

  7. Deficiency of prolactin-inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice.

    PubMed

    Li, Jintao; Liu, Dong; Mou, Zhirong; Ihedioha, Olivia C; Blanchard, Anne; Jia, Ping; Myal, Yvonne; Uzonna, Jude E

    2015-04-01

    Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity. PMID:25594453

  8. Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani.

    PubMed Central

    Chen, M; Christensen, S B; Theander, T G; Kharazmi, A

    1994-01-01

    This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen compared with values for untreated control animals. The [3H]thymidine uptake by the parasites isolated from the treated hamsters was only about 1% of that observed in parasites isolated from the controls. Oral administration of licochalcone A at concentrations of 5 to 150 mg/kg of body weight per day for 6 consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases. PMID:8092835

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

    PubMed

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

    2016-05-01

    Leishmaniases consist of a group of diseases caused by protozoan parasites of Leishmania genus. The outcome of the disease depends on the immune responses of the host as well as the pathogenicity of the strain of the parasite. In murine models, the inoculation of Leishmania major into resistant mice results in Th1 responses and recovery from the infection. However in the susceptible mice, the same inoculation leads to a profile of Th2 responses. Zinc (Zn) is an essential trace element which is required for the growth and development of the immune responses. In this study, the influence of Zn sulfate on mRNA expression of main cytokines of the immune response was studied in susceptible BALB/c mice infected with L. major. The inoculated mice were divided into 3 groups, namely the untreated (control), the zinc sulfate treated (weeks 2, 4 and 8), and the Glucantime-treated (weeks 4 and 8) mice. During different time points post-infection, the lesion sizes and the parasite burden were measured in all the groups. Moreover, the expression of Ifng, Il4, Il10 and Il12 mRNA levels in the draining lymph nodes of the treated mice were compared to the control mice using real-time PCR. Our data demonstrated significant decreases in lesion sizes and parasite loads in Zn sulfate treated group compared to the untreated group. Moreover, significant fold increases in expression of Ifng transcript were observed in mice treated with Zn sulfate compared to the control. The ratio of Ifng/Il4 mRNA was also higher in Zn sulfate-treated mice compared to Glucantime-treated animals. These results indicate that Zn Sulfate has the ability to induce strong Th1 responses in susceptible BALB/c mice inoculated with L. major. PMID:26896749

  10. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  11. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  12. 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. PMID:27068508

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

    PubMed

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

    2016-08-01

    The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states. PMID:27185486

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  16. Critical role of the major histocompatibility complex and IL-10 in matrilin-1-induced relapsing polychondritis in mice.

    PubMed

    Hansson, Ann-Sofie; Johansson, Asa C M; Holmdahl, Rikard

    2004-01-01

    Relapsing polychondritis (RP) is an autoimmune disease that affects extra-articular cartilage. Matrilin-1-induced relapsing polychondritis (MIRP) is a model for RP and is useful for studies of the pathogenic mechanisms in this disease. There are indications that the major histocompatibility complex (MHC) class II plays a major role in RP, since DR4+ patients are more commonly affected than controls. We have now addressed the role of the MHC region, as well as the non-MHC contribution, using congenic mouse strains. Of the MHC congenic strains, B10.Q (H2q) was the most susceptible, the B10.P (H2p) and B10.R (H2r) strains developed mild disease, while B10 strains carrying the v, b, f, or u H2 haplotypes were resistant. A slight variation of susceptibility of H2q strains (B10.Q> C3H.Q> DBA/1) was observed and the (B10.Q x DBA/1)F1 was the most susceptible of all strains. Furthermore, macrophages and CD4+ T cells were the most prominent cell types in inflammatory infiltrates of the tracheal cartilage. Macrophages are the major source of many cytokines, such as interleukin-10 (IL-10), which is currently being tested as a therapeutic agent in several autoimmune diseases. We therefore investigated B10.Q mice devoid of IL-10 through gene deletion and found that they developed a significantly more severe disease, with an earlier onset, than their heterozygous littermates. In conclusion, MHC genes, as well as non-MHC genes, are important for MIRP induction, and IL-10 plays a major suppressive role in cartilage inflammation of the respiratory tract. PMID:15380048

  17. 1H NMR-Based Profiling Reveals Differential Immune-Metabolic Networks during Influenza Virus Infection in Obese Mice

    PubMed Central

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

    2014-01-01

    Obese individuals are at greater risk for death from influenza virus infection. Paralleling human evidence, obese mice are also more susceptible to influenza infection mortality. However, the underlying mechanisms driving greater influenza severity in the obese remain unclear. Metabolic profiling has been utilized in infectious disease models to enhance prognostic or diagnostic methods, and to gain insight into disease pathogenesis by providing a more global picture of dynamic infection responses. Herein, metabolic profiling was used to develop a deeper understanding of the complex processes contributing to impaired influenza protection in obese mice and to facilitate generation of new explanatory hypotheses. Diet-induced obese and lean mice were infected with influenza A/Puerto Rico/8/34. 1H nuclear magnetic resonance-based metabolic profiling of urine, feces, lung, liver, mesenteric white adipose tissue, bronchoalveolar lavage fluid and serum revealed distinct metabolic signatures in infected obese mice, including perturbations in nucleotide, vitamin, ketone body, amino acid, carbohydrate, choline and lipid metabolic pathways. Further, metabolic data was integrated with immune analyses to obtain a more comprehensive understanding of potential immune-metabolic interactions. Of interest, uncovered metabolic signatures in urine and feces allowed for discrimination of infection status in both lean and obese mice at an early influenza time point, which holds prognostic and diagnostic implications for this methodology. These results confirm that obesity causes distinct metabolic perturbations during influenza infection and provide a basis for generation of new hypotheses and use of this methodology in detection of putative biomarkers and metabolic patterns to predict influenza infection outcome. PMID:24844920

  18. Angiography reveals novel features of the retinal vasculature in healthy and diabetic mice.

    PubMed

    McLenachan, Samuel; Magno, Aaron Len; Ramos, David; Catita, Joana; McMenamin, Paul G; Chen, Fred Kuanfu; Rakoczy, Elizabeth Piroska; Ruberte, Jesus

    2015-09-01

    The mouse retina is a commonly used animal model for the study of pathogenesis and treatment of blinding retinal vascular diseases such as diabetic retinopathy. In this study, we aimed to characterize normal and pathological variations in vascular anatomy in the mouse retina using fluorescein angiography visualized with scanning laser ophthalmoscopy and optical coherence tomography (SLO-OCT). We examined eyes from C57BL/6J wild type mice as well as the Ins2(Akita) and Akimba mouse models of diabetic retinopathy using the Heidelberg Retinal Angiography (HRA) and OCT system. Angiography was performed on three focal planes to examine distinct vascular layers. For comparison with angiographic data, ex vivo analyses, including Indian ink angiography, histology and 3D confocal scanning laser microscopy were performed in parallel. All layers of the mouse retinal vasculature could be readily visualized during fluorescein angiography by SLO-OCT. Blood vessel density was increased in the deep vascular plexus (DVP) compared with the superficial vascular plexus (SVP). Arteriolar and venular typologies were established and structural differences were observed between venular types. Unexpectedly, the hyaloid artery was found to persist in 15% of C57BL/6 mice, forming anastomoses with peripheral retinal capillaries. Fluorescein leakage was easily detected in Akimba retinae by angiography, but was not observed in Ins2(Akita) mice. Blood vessel density was increased in the DVP of 6 month old Ins2(Akita) mice, while the SVP displayed reduced branching in precapillary arterioles. In summary, we present the first comprehensive characterization of the mouse retinal vasculature by SLO-OCT fluorescein angiography. Using this clinical imaging technique, we report previously unrecognized variations in C57BL/6J vascular anatomy and novel features of vascular retinopathy in the Ins2(Akita) mouse model of diabetes. PMID:26122048

  19. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    PubMed

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis. PMID:27049463

  20. Differential proteome and gene expression reveal response to carbon ion irradiation in pubertal mice testes.

    PubMed

    Li, Hongyan; He, Yuxuan; Zhang, Hong; Miao, Guoying

    2014-03-21

    Heavy ion radiation, a high linear energy transfer (LET) radiation, has been shown to have adverse effects on reproduction in male mice. The aim of this study was to profile and investigate the differentially expressed proteins in pubertal male mice testes following carbon ion radiation (CIR). Male mice underwent whole-body irradiation with CIR (1 and 4 Gy), and MALDI-TOF/TOF analysis was used to investigate the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 14 days. 8 differentially expressed proteins were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Our results indicated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. PMID:24440814

  1. Modifying Behavioral Phenotypes in Fmr1 KO Mice: Genetic Background Differences Reveal Autistic-Like Responses

    PubMed Central

    Spencer, Corinne M.; Alekseyenko, Olga; Hamilton, Shannon M.; Thomas, Alexia M.; Serysheva, Ekaterina; Yuva-Paylor, Lisa A.; Paylor, Richard

    2010-01-01

    Scientific Abstract Fragile X syndrome (FXS) is the most common inherited form of intellectual disability in humans. In addition to cognitive impairment, patients may exhibit hyperactivity, attention deficits, social difficulties and anxiety, and autistic-like behaviors. The degree to which patients display these behaviors varies considerably and is influenced by family history, suggesting that genetic modifiers play a role in the expression of behaviors in FXS. Several studies have examined behavior in a mouse model of FXS in which the Fmr1 gene has been ablated. Most of those studies were done in Fmr1 knockout mice on a pure C57BL/6 or FVB strain background. To gain a better understanding of the effects of genetic background on behaviors resulting from the loss of Fmr1 gene expression, we generated F1 hybrid lines from female Fmr1 heterozygous mice on a pure C57BL/6J background bred with male Fmr1 wild-type mice of various background strains (A/J, DBA/2J, FVB/NJ, 129S1/SvImJ and CD-1). Male Fmr1 knockout and wild-type littermates from each line were examined in an extensive behavioral test battery. Results clearly indicate that multiple behavioral responses are dependent on genetic background, including autistic-like traits that are present on limited genetic backgrounds. This approach has allowed us to identify improved models for different behavioral symptoms present in FXS including autistic-like traits. PMID:21268289

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

    PubMed Central

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

    2015-01-01

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

  3. 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

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

    PubMed

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

    2008-09-23

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

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

    PubMed Central

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

    2008-01-01

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

  6. 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

    2012-01-01

    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

  7. Vaccination with phosphoglycan-deficient Leishmania major protects highly susceptible mice from virulent challenge without inducing a strong Th1 response.

    PubMed

    Uzonna, Jude E; Späth, Gerald F; Beverley, Stephen M; Scott, Phillip

    2004-03-15

    Long-term immunity to Leishmania may require the continued presence of parasites, but previous attempts to create attenuated parasites that persist without causing disease have had limited success. Since Leishmania major mutants that lack lipophosphoglycan and other secreted phosphoglycans, termed lpg2-, persist indefinitely in infected mice without inducing any disease, we tested their ability to provide protection to virulent L. major challenge. In response to leishmanial Ag stimulation, cells from lpg2--infected mice produced minimal levels of IL-4 and IL-10, as well as very low levels of IFN-gamma. Nevertheless, when BALB/c mice infected with lpg2- parasites were challenged with virulent L. major they were protected from disease. Thus, these findings report on attenuated parasites that may be used to induce long-term protection against leishmaniasis and indicate that the immunity induced can be maintained in the absence of a strong Th1 response. PMID:15004184

  8. Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window.

    PubMed

    Polizzotti, Brian D; Ganapathy, Balakrishnan; Walsh, Stuart; Choudhury, Sangita; Ammanamanchi, Niyatie; Bennett, David G; dos Remedios, Cristobal G; Haubner, Bernhard J; Penninger, Josef M; Kühn, Bernhard

    2015-04-01

    Therapies developed for adult patients with heart failure have been shown to be ineffective in pediatric clinical trials, leading to the recognition that new pediatric-specific therapies for heart failure must be developed. Administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates regeneration of heart muscle cells (cardiomyocytes) in adult mice. Because proliferation-competent cardiomyocytes are more abundant in growing mammals, we hypothesized that administration of rNRG1 during the neonatal period might be more effective than in adulthood. If so, neonatal rNRG1 delivery could be a new therapeutic strategy for treating heart failure in pediatric patients. To evaluate the effectiveness of rNRG1 administration in cardiac regeneration, newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity. Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars. In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). We also assessed the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease who were less than 6 months of age. Our results identify an effective time period within which to execute rNRG1 clinical trials in pediatric patients for the stimulation of cardiomyocyte regeneration. PMID:25834111

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

    PubMed Central

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

    2016-01-01

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

  10. 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. PMID:26707637

  11. Characterizing VIP Neurons in the Barrel Cortex of VIPcre/tdTomato Mice Reveals Layer-Specific Differences

    PubMed Central

    Prönneke, Alvar; Scheuer, Bianca; Wagener, Robin J.; Möck, Martin; Witte, Mirko; Staiger, Jochen F.

    2015-01-01

    Neocortical GABAergic interneurons have a profound impact on cortical circuitry and its information processing capacity. Distinct subgroups of inhibitory interneurons can be distinguished by molecular markers, such as parvalbumin, somatostatin, and vasoactive intestinal polypeptide (VIP). Among these, VIP-expressing interneurons sparked a substantial interest since these neurons seem to operate disinhibitory circuit motifs found in all major neocortical areas. Several of these recent studies used transgenic Vip-ires-cre mice to specifically target the population of VIP-expressing interneurons. This makes it necessary to elucidate in detail the sensitivity and specificity of Cre expression for VIP neurons in these animals. Thus, we quantitatively compared endogenous tdTomato with Vip fluorescence in situ hybridization and αVIP immunohistochemistry in the barrel cortex of VIPcre/tdTomato mice in a layer-specific manner. We show that VIPcre/tdTomato mice are highly sensitive and specific for the entire population of VIP-expressing neurons. In the barrel cortex, approximately 13% of all GABAergic neurons are VIP expressing. Most VIP neurons are found in layer II/III (∼60%), whereas approximately 40% are found in the other layers of the barrel cortex. Layer II/III VIP neurons are significantly different from VIP neurons in layers IV–VI in several morphological and membrane properties, which suggest layer-dependent differences in functionality. PMID:26420784

  12. Shotgun proteomics reveals possible mechanisms for cognitive impairment in Mucopolysaccharidosis I mice.

    PubMed

    Baldo, Guilherme; Lorenzini, Daniel Macedo; Santos, Diogenes Santiago; Mayer, Fabiana Quoos; Vitry, Sandrine; Bigou, Stephanie; Heard, Jean Michael; Matte, Ursula; Giugliani, Roberto

    2015-02-01

    Mucopolysaccharidosis type I (MPS I) is due to deficient alpha-L-iduronidase (IDUA) which leads to storage of undegraded glycosaminoglycans (GAG). The severe form of the disease is characterized by mental retardation of unknown etiology. Trying to unveil the mechanisms that lead to cognitive impairment in MPS I, we studied alterations in the proteome from MPS I mouse hippocampus. Eight-month old mice presented increased LAMP-1 expression, GAG storage in neurons and glial cells, and impaired aversive and non-aversive memory. Shotgun proteomics was performed and 297 proteins were identified. Of those, 32 were differentially expressed. We found elevation in proteins such as cathepsins B and D; however their increase did not lead to cell death in MPS I brains. Glial fibrillary acid protein (GFAP) was markedly elevated, and immunohistochemistry confirmed a neuroinflammatory process that could be responsible for neuronal dysfunction. We didn't observe any differences in ubiquitin expression, as well as in other proteins related to protein folding, suggesting that the ubiquitin system is working properly. Finally, we observed alterations in several proteins involved in synaptic plasticity, including overexpression of post synaptic density-95 (PSD95) and reduction of microtubule-associated proteins 1A and 1B. These results together suggest that the cognitive impairment in MPS I mice is not due to massive cell death, but rather to neuronal dysfunction caused by multiple processes, including neuroinflammation and alterations in synaptic plasticity. PMID:25541102

  13. 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. PMID:21177255

  14. 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

  15. 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

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

    PubMed

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

    2016-03-22

    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

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

    PubMed

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

    2016-07-01

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

  18. Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula.

    PubMed

    Camps, Céline; Jardinaud, Marie-Françoise; Rengel, David; Carrère, Sébastien; Hervé, Christine; Debellé, Frédéric; Gamas, Pascal; Bensmihen, Sandra; Gough, Clare

    2015-10-01

    Myc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control. Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated. Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3- and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3- and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors. These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP. PMID:25919491

  19. 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

  20. 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...

  1. Sodium Iodide Symporter PET and BLI Noninvasively Reveal Mesoangioblast Survival in Dystrophic Mice.

    PubMed

    Holvoet, Bryan; Quattrocelli, Mattia; Belderbos, Sarah; Pollaris, Lore; Wolfs, Esther; Gheysens, Olivier; Gijsbers, Rik; Vanoirbeek, Jeroen; Verfaillie, Catherine M; Sampaolesi, Maurilio; Deroose, Christophe M

    2015-12-01

    Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells. PMID:26626179

  2. Sodium Iodide Symporter PET and BLI Noninvasively Reveal Mesoangioblast Survival in Dystrophic Mice

    PubMed Central

    Holvoet, Bryan; Quattrocelli, Mattia; Belderbos, Sarah; Pollaris, Lore; Wolfs, Esther; Gheysens, Olivier; Gijsbers, Rik; Vanoirbeek, Jeroen; Verfaillie, Catherine M.; Sampaolesi, Maurilio; Deroose, Christophe M.

    2015-01-01

    Summary Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells. PMID:26626179

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

    PubMed Central

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

    2015-01-01

    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 regulating 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 neurodevelopmental and other psychiatric conditions. PMID:26066729

  4. Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy

    PubMed Central

    Giordano, Christian; Mojumdar, Kamalika; Liang, Feng; Lemaire, Christian; Li, Tong; Richardson, John; Divangahi, Maziar; Qureshi, Salman; Petrof, Basil J.

    2015-01-01

    Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOSneg CD206pos) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1–TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression. PMID:25552658

  5. 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

  6. 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

  7. Expression profiling reveals novel hypoxic biomarkers in peripheral blood of adult mice exposed to chronic hypoxia.

    PubMed

    Mosqueira, Matias; Willmann, Gabriel; 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

  8. 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

  9. Comparison of RELMα and RELMβ Single- and Double-Gene-Deficient Mice Reveals that RELMα Expression Dictates Inflammation and Worm Expulsion in Hookworm Infection.

    PubMed

    Chen, Gang; Wang, Spencer H; Jang, Jessica C; Odegaard, Justin I; Nair, Meera G

    2016-04-01

    Resistin-like molecules (RELMs) are highly expressed following helminth infection, where they impact both the host and helminth. While RELMα (Retnla) impairs helminth expulsion by inhibiting protective Th2 immunity, RELMβ (Retnlb) can promote its expulsion. We employed Retnla(-/-) and Retnlb(-/-) mice to delineate the function of both proteins following infection with Nippostrongylus brasiliensis, a hookworm that infects the lung and intestine. Whereas wild-type (WT) and Retnlb(-/-)mice exhibited equivalent infection-induced inflammation, Retnla(-/-) mice suffered a heightened inflammatory response, including increased mortality, weight loss, and lung inflammation. In the intestine, Retnla(-/-)mice had low parasite egg burdens compared to those of WT mice, while Retnlb(-/-) mice exhibited high egg burdens, suggesting that RELMα and RELMβ have functionally distinct effects on immunity and inflammation to N. brasiliensis To test the importance of both proteins, we generated Retnla(-/-) Retnlb(-/-) mice. Infected Retnla(-/-)Retnlb(-/-) mice exhibited similar responses to Retnla(-/-) mice, including increased mortality and lung inflammation. This inflammatory response in Retnla(-/-) Retnlb(-/-) mice negatively impacted N. brasiliensis fitness, as demonstrated by significantly lower worm ATP levels and decreased intestinal worm burden and fecundity. Lung cytokine analysis revealed that Retnla(-/-) and Retnla(-/-) Retnlb(-/-) mice expressed significantly increased levels of interleukin-4 (IL-4). Finally, we generated Retnla(-/-) mice on the Rag(-/-) background and observed that the effects of RELMα were abrogated in the absence of adaptive immunity. Together, these data demonstrate that RELMα but not RELMβ significantly impacts the immune response toN. brasiliensis infection by downregulating the Th2 adaptive immune response in the lung, which protects the host but allows improved parasite fitness. PMID:26831469

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

    PubMed Central

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

    2013-01-01

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

  11. Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells.

    PubMed

    Yang, Jianchang; Chai, Li; Fowles, Taylor C; Alipio, Zaida; Xu, Dan; Fink, Louis M; Ward, David C; Ma, Yupo

    2008-12-16

    Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls. PMID:19060217

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

    PubMed

    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 conditions

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

    PubMed Central

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

    2005-01-01

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

  14. 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

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

    PubMed Central

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

    2010-01-01

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

  16. Aromatase knockout mice reveal an impact of estrogen on drug-induced alternation of murine electrocardiography parameters.

    PubMed

    Kurokawa, Junko; Sasano, Tetsuo; Kodama, Masami; Li, Min; Ebana, Yusuke; Harada, Nobuhiro; Honda, Shin-ichiro; Nakaya, Haruaki; Furukawa, Tetsushi

    2015-06-01

    Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology. PMID:25972195

  17. 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

  18. Disease-Drug Pairs Revealed by Computational Genomic Connectivity Mapping on GBA1 Deficient, Gaucher Disease Mice

    PubMed Central

    Yuen, Tony; Iqbal, Jameel; Zhu, Ling-Ling; Sun, Li; Lin, Aiping; Zhao, Hongyu; Liu, Jun; Mistry, Pramod K.; Zaidi, Mone

    2012-01-01

    We have reported that, in addition to recapitulating the classical human Gaucher disease (GD1) phenotype, deletion of the glucocerebrosidase (GBA1) gene in mice results in the dysfunction of a diverse population of immune cells. Most of immune-related, non-classical features of GD1, including gammopathies and autoimmune diathesis, are resistant to macrophage-directed therapies. This has prompted a search for newer agents for human GD1. Here, we used high-density microarray on splenic and liver cells from affected GBA1−/− mice to establish a gene “signature”, which was then utilized to interrogate the Broad Institute database, CMAP. Computational connectivity mapping of disease and drug pairs through CMAP revealed several highly enriched, non-null, mimic and anti-mimic hits. Most notably, two compounds with anti-helminthic properties, namely albendazole and oxamniquine, were identified; these are particularly relevant for future testing as the expression of chitinases is enhanced in GD1. PMID:22588172

  19. Toxicogenomic analysis of mainstream tobacco smoke-exposed mice reveals repression of plasminogen activator inhibitor-1 gene in heart.

    PubMed

    Halappanavar, Sabina; Stampfli, Martin R; Berndt-Weis, Lynn; Williams, Andrew; Douglas, George R; Yauk, Carole L

    2009-01-01

    Tobacco smoking is associated with cardiovascular pathology. However, the molecular mechanisms of tobacco smoke exposure that lead to initiation or exacerbation of cardiovascular disease are unclear. In this study, the effects of mainstream tobacco smoke (MTS) on global transcription in the heart were investigated. Male C57B1/CBA mice were exposed to MTS from 2 cigarettes daily, 5 days/wk for 6 or 12 wk. Mice were sacrificed immediately, or 6 wk following the last cigarette. High-density DNA microarrays were used to characterize global gene expression changes in whole heart. Fifteen genes were significantly differentially expressed following exposure to MTS. Among these genes, cytochrome P-450 1A1 (Cyp1A1) was upregulated by 12-fold, and Serpine-1 (plasminogen activator inhibitor-1, PAI-1) was downregulated by 1.7-fold. Concomitant increase in Cyp1A1 protein levels and decrease in total and active PAI-1 protein was observed in tissue extracts by Western blot assay and enzyme-linked immunosorbent assay (ELISA), respectively. Observed changes were transient and were partially reversed during break periods. Thus, gene expression profiling of heart tissue revealed a novel cardiovascular mechanism operating in response to MTS. Our results suggest a potential role for PAI-1 in MTS-induced cardiovascular pathology. PMID:18925475

  20. 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

  1. Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model.

    PubMed

    Katebi, A; Gholami, E; Taheri, T; Zahedifard, F; Habibzadeh, S; Taslimi, Y; Shokri, F; Papadopoulou, B; Kamhawi, S; Valenzuela, J G; Rafati, S

    2015-10-01

    Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis. PMID:26298575

  2. Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

    PubMed Central

    White, Jacqueline K.; Gerdin, Anna-Karin; Karp, Natasha A.; Ryder, Ed; Buljan, Marija; Bussell, James N.; Salisbury, Jennifer; Clare, Simon; Ingham, Neil J.; Podrini, Christine; Houghton, Richard; Estabel, Jeanne; Bottomley, Joanna R.; Melvin, David G.; Sunter, David; Adams, Niels C.; Baker, Lauren; Barnes, Caroline; Beveridge, Ryan; Cambridge, Emma; Carragher, Damian; Chana, Prabhjoat; Clarke, Kay; Hooks, Yvette; Igosheva, Natalia; Ismail, Ozama; Jackson, Hannah; Kane, Leanne; Lacey, Rosalind; Lafont, David Tino; Lucas, Mark; Maguire, Simon; McGill, Katherine; McIntyre, Rebecca E.; Messager, Sophie; Mottram, Lynda; Mulderrig, Lee; Pearson, Selina; Protheroe, Hayley J.; Roberson, Laura-Anne; Salsbury, Grace; Sanderson, Mark; Sanger, Daniel; Shannon, Carl; Thompson, Paul C.; Tuck, Elizabeth; Vancollie, Valerie E.; Brackenbury, Lisa; Bushell, Wendy; Cook, Ross; Dalvi, Priya; Gleeson, Diane; Habib, Bishoy; Hardy, Matt; Liakath-Ali, Kifayathullah; Miklejewska, Evelina; Price, Stacey; Sethi, Debarati; Trenchard, Elizabeth; von Schiller, Dominique; Vyas, Sapna; West, Anthony P.; Woodward, John; Wynn, Elizabeth; Evans, Arthur; Gannon, David; Griffiths, Mark; Holroyd, Simon; Iyer, Vivek; Kipp, Christian; Lewis, Morag; Li, Wei; Oakley, Darren; Richardson, David; Smedley, Damian; Agu, Chukwuma; Bryant, Jackie; Delaney, Liz; Gueorguieva, Nadia I.; Tharagonnet, Helen; Townsend, Anne J.; Biggs, Daniel; Brown, Ellen; Collinson, Adam; Dumeau, Charles-Etienne; Grau, Evelyn; Harrison, Sarah; Harrison, James; Ingle, Catherine E.; Kundi, Helen; Madich, Alla; Mayhew, Danielle; Metcalf, Tom; Newman, Stuart; Pass, Johanna; Pearson, Laila; Reynolds, Helen; Sinclair, Caroline; Wardle-Jones, Hannah; Woods, Michael; Alexander, Liam; Brown, Terry; Flack, Francesca; Frost, Carole; Griggs, Nicola; Hrnciarova, Silvia; Kirton, Andrea; McDermott, Jordan; Rogerson, Claire; White, Gemma; Zielezinski, Pawel; DiTommaso, Tia; Edwards, Andrew; Heath, Emma; Mahajan, Mary Ann; Yalcin, Binnaz; Tannahill, David; Logan, Darren W.; MacArthur, Daniel G.; Flint, Jonathan; Mahajan, Vinit B.; Tsang, Stephen H.; Smyth, Ian; Watt, Fiona M.; Skarnes, William C.; Dougan, Gordon; Adams, David J.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.

    2013-01-01

    Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip PMID:23870131

  3. Direct comparison of mice null for liver or intestinal fatty acid-binding proteins reveals highly divergent phenotypic responses to high fat feeding.

    PubMed

    Gajda, Angela M; Zhou, Yin Xiu; Agellon, Luis B; Fried, Susan K; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-10-18

    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

  4. Controlled DNA double-strand break induction in mice reveals post-damage transcriptome stability.

    PubMed

    Kim, Jeongkyu; Sturgill, David; Tran, Andy D; Sinclair, David A; Oberdoerffer, Philipp

    2016-04-20

    DNA double-strand breaks (DSBs) and their repair can cause extensive epigenetic changes. As a result, DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways. Studying the consequences of DSBs in higher organisms has, however, been hindered by a scarcity of tools for controlled DSB induction. Here, we describe a mouse model that allows for both tissue-specific and temporally controlled DSB formation at ∼140 defined genomic loci. Using this model, we show that DSBs promote a DNA damage signaling-dependent decrease in gene expression in primary cells specifically at break-bearing genes, which is reversed upon DSB repair. Importantly, we demonstrate that restoration of gene expression can occur independently of cell cycle progression, underlining its relevance for normal tissue maintenance. Consistent with this, we observe no evidence for persistent transcriptional repression in response to a multi-day course of continuous DSB formation and repair in mouse lymphocytesin 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. PMID:26687720

  5. Controlled DNA double-strand break induction in mice reveals post-damage transcriptome stability

    PubMed Central

    Kim, Jeongkyu; Sturgill, David; Tran, Andy D.; Sinclair, David A.; Oberdoerffer, Philipp

    2016-01-01

    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. PMID:26687720

  6. Immunostimulatory responses to crude extracts of Warburgia ugandensis (sprague) subsp ugandensis (canellaceae) by Balb/c mice infected with Leishmania major

    PubMed Central

    Ngure, Peter; Ng'ang'a, Zipporah; Kimutai, Albert; Kepha, Stella; Mong'are, Samuel; Ingonga, Johnnie; Tonui, Willy

    2014-01-01

    Introduction To determine the immunostimulatory potential of crude extracts of Warburgia ugandensis subsp. ugandensis with a soluble leishmanial antigen in vaccinating BALB/c mice. Methods Seventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble leishmania antigens (SLA) alone, hexane, ethyl acetate, and dichloromethane extract co-administered with SLA. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad. Results On vaccination with extracts of W. ugandensis subsp. ugandensis alone or as adjuvants when used in combination with Leishmania antigens, the hexane extract and the dichloromethane extract plus SLA stimulated moderate production of IFN-γ and low levels of IL-4.These mice were partially protected from cutaneous leishmaniasis as shown by the slow development of lesions and comparatively less parasite burdens. Conclusion These data suggest that extracts of W. ugandensis subsp. ugandensis are suitable adjuvants for Leishmania vaccines. However, since W. ugandensis subsp. ugandensis has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with a soluble leishmanial antigen in vaccinating BALB/c mice. PMID:24624248

  7. Quantitative genetic study of maximal electroshock seizure threshold in mice: evidence for a major seizure susceptibility locus on distal chromosome 1.

    PubMed

    Ferraro, T N; Golden, G T; Smith, G G; Longman, R L; Snyder, R L; DeMuth, D; Szpilzak, I; Mulholland, N; Eng, E; Lohoff, F W; Buono, R J; Berrettini, W H

    2001-07-01

    We conducted a quantitative trait locus (QTL) mapping study to dissect the multifactorial nature of maximal electroshock seizure threshold (MEST) in C57BL/6 (B6) and DBA/2 (D2) mice. MEST determination involved a standard paradigm in which 8- to 12-week-old mice received one shock per day with a daily incremental increase in electrical current until a maximal seizure (tonic hindlimb extension) was induced. Mean MEST values in parental strains were separated by over five standard deviation units, with D2 mice showing lower values than B6 mice. The distribution of MEST values in B6xD2 F2 intercrossed mice spanned the entire phenotypic range defined by parental strains. Statistical mapping yielded significant evidence for QTLs on chromosomes 1, 2, 5, and 15, which together explained over 60% of the phenotypic variance in the model. The chromosome 1 QTL represents a locus of major effect, accounting for about one-third of the genetic variance. Experiments involving a congenic strain (B6.D2-Mtv7(a)/Ty) enabled more precise mapping of the chromosome 1 QTL and indicate that it lies in the genetic interval between markers D1Mit145 and D1Mit17. These results support the hypothesis that the distal portion of chromosome 1 harbors a gene(s) that has a fundamental role in regulating seizure susceptibility. PMID:11472065

  8. 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.

  9. Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

    PubMed Central

    Rivera, Mercedes F.; Lee, Ju-Youn; Aneja, Monika; Goswami, Vishalkant; Liu, Liying; Velsko, Irina M.; Chukkapalli, Sasanka S.; Bhattacharyya, Indraneel; Chen, Hao; Lucas, Alexandra R.; Kesavalu, Lakshmyya N.

    2013-01-01

    Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoEnull) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoEnull mice. PMID:23451182

  10. Urinary Metabolomics Reveals Alterations of Aromatic Amino Acid Metabolism of Alzheimer's Disease in the Transgenic CRND8 Mice.

    PubMed

    Tang, Zhi; Liu, Liangfeng; Li, Yongle; Dong, Jiyang; Li, Min; Huang, Jiandong; Lin, Shuhai; Cai, Zongwei

    2016-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with amyloid plaques accumulation as the key feature involved in its pathology. To date, however, the biochemical changes in AD have not been clearly characterized. Here, we present that urinary metabolomics based on high resolution mass spectrometry was employed for delineation of metabolic alterations in transgenic CRND8 mice. In this noninvasive approach, urinary metabolome reveals the biochemical changes in early onset of this AD mouse model. In virtue of comprehensive metabolite profiling and multivariate statistical analysis, a total of 73 differential metabolites of urine sample sets was identified in 12-week and 18-week transgenic mice compared to wild-type littermates, covering perturbations of aromatic amino acid metabolism, the Krebs cycle and one-carbon metabolism. Of particular interest is that divergent tryptophan metabolism, such as upregulation of serotonin pathway while downregulation of kynurenine pathway, was observed. Meanwhile, the accumulation of both N-acetylvanilalanine and 3-methoxytyrosine indicated aromatic L-amino acid decarboxylase deficiency. And the microbial metabolites derived from aromatic amino acid metabolism and drug-like phase II metabolic response via the glycine conjugation reactions were also highlighted, indicating that genetic modification in mouse brain not only alters genotype but also perturbs the gut microbiome. Together, our study demonstrated that the integrative approach employing mass spectrometry-based metabolomics and a transgenic mouse model for AD may provide new evidence for distinct metabolic signatures. The perturbations of metabolic pathways may have far-reaching implications for early diagnosis and intervention in AD. PMID:26825095

  11. Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

    PubMed Central

    Chelh, Ilham; Meunier, Bruno; Picard, Brigitte; Reecy, Mark James; Chevalier, Catherine; Hocquette, Jean-François; Cassar-Malek, Isabelle

    2009-01-01

    Background Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. Results Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. Conclusion All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3ε protein, TCTP/GSK-3β). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo. PMID:19397818

  12. 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

  13. Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice1

    PubMed Central

    Yu, Hong; Karunakaran, Karuna P.; Jiang, Xiaozhou; Brunham, Robert C.

    2014-01-01

    An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of C. muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population. PMID:24992718

  14. Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

    PubMed Central

    Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

    2014-01-01

    Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

  15. 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. PMID:25929334

  16. Molecular Phenotyping of Immune Cells from Young NOD Mice Reveals Abnormal Metabolic Pathways in the Early Induction Phase of Autoimmune Diabetes

    PubMed Central

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

    2012-01-01

    Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse – a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (∼90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes. PMID:23071669

  17. 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. PMID:23071669

  18. Evaluation of recombinant Leishmania poly-protein plus GLA-SE vaccines against sand fly-transmitted Leishmania major in C57Bl/6 mice1

    PubMed Central

    Peters, Nathan C.; Bertholet, Sylvie; Lawyer, Phillip G.; Charmoy, Melanie; Romano, Audrey; Ribeiro-Gomes, Flavia L.; Stamper, Lisa W.; Sacks, David L.

    2012-01-01

    Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania poly-proteins delivered in a stable emulsion (SE) with the TLR 4 agonists monophosphoryl lipid A (MPL) or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of L. major in susceptible BALB/c mice. Similar poly-protein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Poly-protein vaccinated mice had a 60-fold increase in CD4+IFN-γ+ T cell numbers versus control animals at 2 weeks post needle inoculation of L. major and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, poly-protein vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+ IFN-γ+:IL-17+ ratios versus non-vaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4+ IFN-γ+:IL-17+ ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite. PMID:23045616

  19. 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

  20. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice

    PubMed Central

    Shiao, Young-Ji; Liou, Kuo-Tong; Hsu, Wei-Hsiang; Hsieh, Pei-Hsuan; Lee, Chi-Ying; Chen, Yet-Ran; Lin, Yun-Lian

    2015-01-01

    Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood–brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke. PMID:26492191

  1. 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

  2. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

    PubMed

    Chen, Hong-Jhang; Shen, Yuh-Chiang; Shiao, Young-Ji; Liou, Kuo-Tong; Hsu, Wei-Hsiang; Hsieh, Pei-Hsuan; Lee, Chi-Ying; Chen, Yet-Ran; Lin, Yun-Lian

    2015-01-01

    Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke. PMID:26492191

  3. Major mouse placental compartments revealed by diffusion-weighted MRI, contrast-enhanced MRI, and fluorescence imaging

    PubMed Central

    Solomon, Eddy; Avni, Reut; Hadas, Ron; Raz, Tal; Garbow, Joel Richard; Bendel, Peter; Frydman, Lucio; Neeman, Michal

    2014-01-01

    Mammalian models, and mouse studies in particular, play a central role in our understanding of placental development. Magnetic resonance imaging (MRI) could be a valuable tool to further these studies, providing both structural and functional information. As fluid dynamics throughout the placenta are driven by a variety of flow and diffusion processes, diffusion-weighted MRI could enhance our understanding of the exchange properties of maternal and fetal blood pools—and thereby of placental function. These studies, however, have so far been hindered by the small sizes, the unavoidable motions, and the challenging air/water/fat heterogeneities, associated with mouse placental environments. The present study demonstrates that emerging methods based on the spatiotemporal encoding (SPEN) of the MRI information can robustly overcome these obstacles. Using SPEN MRI in combination with albumin-based contrast agents, we analyzed the diffusion behavior of developing placentas in a cohort of mice. These studies successfully discriminated the maternal from the fetal blood flows; the two orders of magnitude differences measured in these fluids’ apparent diffusion coefficients suggest a nearly free diffusion behavior for the former and a strong flow-based component for the latter. An intermediate behavior was observed by these methods for a third compartment that, based on maternal albumin endocytosis, was associated with trophoblastic cells in the interphase labyrinth. Structural features associated with these dynamic measurements were consistent with independent intravital and ex vivo fluorescence microscopy studies and are discussed within the context of the anatomy of developing mouse placentas. PMID:24969421

  4. Major mouse placental compartments revealed by diffusion-weighted MRI, contrast-enhanced MRI, and fluorescence imaging.

    PubMed

    Solomon, Eddy; Avni, Reut; Hadas, Ron; Raz, Tal; Garbow, Joel Richard; Bendel, Peter; Frydman, Lucio; Neeman, Michal

    2014-07-15

    Mammalian models, and mouse studies in particular, play a central role in our understanding of placental development. Magnetic resonance imaging (MRI) could be a valuable tool to further these studies, providing both structural and functional information. As fluid dynamics throughout the placenta are driven by a variety of flow and diffusion processes, diffusion-weighted MRI could enhance our understanding of the exchange properties of maternal and fetal blood pools--and thereby of placental function. These studies, however, have so far been hindered by the small sizes, the unavoidable motions, and the challenging air/water/fat heterogeneities, associated with mouse placental environments. The present study demonstrates that emerging methods based on the spatiotemporal encoding (SPEN) of the MRI information can robustly overcome these obstacles. Using SPEN MRI in combination with albumin-based contrast agents, we analyzed the diffusion behavior of developing placentas in a cohort of mice. These studies successfully discriminated the maternal from the fetal blood flows; the two orders of magnitude differences measured in these fluids' apparent diffusion coefficients suggest a nearly free diffusion behavior for the former and a strong flow-based component for the latter. An intermediate behavior was observed by these methods for a third compartment that, based on maternal albumin endocytosis, was associated with trophoblastic cells in the interphase labyrinth. Structural features associated with these dynamic measurements were consistent with independent intravital and ex vivo fluorescence microscopy studies and are discussed within the context of the anatomy of developing mouse placentas. PMID:24969421

  5. Polymerase chain reaction detection and inducible nitric-oxide synthase expression of Leishmania major in mice inoculated by two different routes

    PubMed Central

    Mahmoud, Abeer E; Attia, Rasha AH; Eldeek, Hanan EM; Farrag, Haiam Mohammed Mahmoud; Makboul, Rania

    2016-01-01

    Introduction: Leishmania major needs a sensitive and specific method for proper diagnosis. This study aims to study the course and histopathology of L. major in certain tissues of experimentally infected BALB/c mice after subcutaneous (sc) and intraperitoneal (ip) inoculation. Materials and Methods: After infecting BALB/c mice using sc and ip inoculation, the histopathology was studied. The kinetoplastic DNA polymerase chain reaction (PCR) for its molecular detection and detect the inducible nitric-oxide synthase (iNOS) pattern during the first 3 months of infection. Result: PCR could detect the presence of L. major in all spleens, lymph nodes, and skin ulcers by both inoculation routes while (33%) and (42%) of livers were positive after sc and ip routes, respectively. Chronic inflammatory cell infiltrates with capsulitis was found in the spleen, lymph nodes, and liver. Granulomas were found in the spleen and liver. There was a statistically significant difference in iNOS expression along the experiment in the spleen and lymph nodes by both routes and in the liver by ip only. Apart from the liver, iNOS could not be detected on the 2nd week postinfection and was high after 1 month for both routes in all samples; a moderate decrease at 2 months and the highest decrease were detected after 3 months. Conclusions: L. major inoculation by both routes produce visceral disease in mice, and kinetoplastic DNA PCR can detect its presence from the 2nd week up to the 3rd month postinfection. The iNOS expression was high at 1 and 2 months and remained throughout the 3 months of the experiment; which plays an important role in the disease course and control. PMID:26998433

  6. 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. PMID:20048751

  7. Effects of repeated adolescent stress and serotonin transporter gene partial knockout in mice on behaviors and brain structures relevant to major depression

    PubMed Central

    Spinelli, Simona; Müller, Tanja; Friedel, Miriam; Sigrist, Hannes; Lesch, Klaus-Peter; Henkelman, Mark; Rudin, Markus; Seifritz, Erich; Pryce, Christopher R.

    2013-01-01

    In humans, exposure to stress during development is associated with structural and functional alterations of the prefrontal cortex (PFC), amygdala (AMY), and hippocampus (HC) and their circuits of connectivity, and with an increased risk for developing major depressive disorder particularly in carriers of the short (s) variant of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR). Although changes in these regions are found in carriers of the s allele and/or in depressed patients, evidence for a specific genotype × developmental stress effect on brain structure and function is limited. Here, we investigated the effect of repeated stress exposure during adolescence in mice with partial knockout of the 5-HTT gene (HET) vs. wildtype (WT) on early-adulthood behavioral measures and brain structure [using magnetic resonance imaging (MRI)] relevant to human major depression. Behaviorally, adolescent stress (AS) increased anxiety and decreased activity and did so to a similar degree in HET and WT. In a probabilistic reversal learning task, HET-AS mice achieved fewer reversals than did HET-No-AS mice. 5-HTT genotype and AS were without effect on corticosterone stress response. In terms of structural brain differences, AS reduced the volume of two long-range white matter tracts, the optic tract (OT) and the cerebral peduncle (CP), in WT mice specifically. In a region-of-interest analysis, AS was associated with increased HC volume and HET genotype with a decreased frontal lobe volume. In conclusion, we found that 5-HTT and AS genotype exerted long-term effects on behavior and development of brain regions relevant to human depression. PMID:24427124

  8. Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding.

    PubMed

    Zhang, Ying; Muller, Markus; Xu, Bo; Yoshida, Yutaka; Horlacher, Oliver; Nikitin, Frederic; Garessus, Samuel; Magdeldin, Sameh; Kinoshita, Naohiko; Fujinaka, Hidehiko; Yaoita, Eishin; Hasegawa, Miki; Lisacek, Frederique; Yamamoto, Tadashi

    2015-08-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar. PMID:25825003

  9. 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

  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. 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.

  12. 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. PMID:25989417

  13. Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: Evidence for the existence of two major genotypes.

    PubMed

    Ye, Chao; Zhang, Qing-Zhan; Tian, Zhi-Jun; Zheng, Hao; Zhao, Kuan; Liu, Fei; Guo, Jin-Chao; Tong, Wu; Jiang, Cheng-Gang; Wang, Shu-Jie; Shi, Mang; Chang, Xiao-Bo; Jiang, Yi-Feng; Peng, Jin-Mei; Zhou, Yan-Jun; Tang, Yan-Dong; Sun, Ming-Xia; Cai, Xue-Hui; An, Tong-Qing; Tong, Guang-Zhi

    2015-09-01

    Recently pseudorabies outbreaks have occurred in many vaccinated farms in China. To identify genetic characteristics of pseudorabies virus (PRV) strains, we obtained the genomic sequences of PRV strains HeN1 and JS, which were compared to 4 PRV genomes and 729 partial gene sequences. PRV strains isolated in China showed marked sequence divergence compared to European and American strains. Phylogenetic analysis revealed that for the first time PRV can be divided into 2 distinct clusters, with Chinese strains being genotype II and PRVs isolated from other countries being genotype I. Restriction fragment length polymorphism analysis confirmed differences between HeN1 and Bartha strains, as did the presence of unique insertion/deletion polymorphisms and microsatellites. This divergence between the two genotypes may have been generated from long-term, independent evolution, which could also explain the low efficacy of the Bartha vaccine in protecting pigs infected with genotype II PRV. PMID:25965793

  14. 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

  15. Temporal correlation patterns in pre-seismic electromagnetic emissions reveal distinct complexity profiles prior to major earthquakes

    NASA Astrophysics Data System (ADS)

    Donner, Reik V.; Potirakis, Stelios M.; Balasis, George; Eftaxias, Konstantinos; Kurths, Jürgen

    2015-04-01

    In the last years, continuous recordings of electromagnetic emissions from geophysical observatories have been recognized to exhibit characteristic fluctuation patterns prior to some major earthquakes. To further evaluate and quantify these findings, this work presents a detailed assessment of the time-varying correlation properties of such emissions during the preparatory phases preceding some recent earthquakes in Greece and Italy. During certain stages before the earthquakes' occurrences, the electromagnetic variability profiles are characterized by a marked increase in the degree of organization of fluctuations, which allow developing hypotheses about the underlying physical mechanisms. Based on the preparatory phases of selected seismic events, the information provided by different statistical properties characterizing complementary aspects of the time-varying complexity based on temporal correlations is systematically assessed. The obtained results allow further insights into different pre-seismic stages based on the variability of electromagnetic emissions, which are probably associated with distinct geophysical processes.

  16. 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. PMID:26328757

  17. Phylogeny of the monarch flycatchers reveals extensive paraphyly and novel relationships within a major Australo-Pacific radiation.

    PubMed

    Andersen, Michael J; Hosner, Peter A; Filardi, Christopher E; Moyle, Robert G

    2015-02-01

    Monarch flycatchers are a major component of Australo-Pacific and Wallacean avifaunas. To date, the family has received incomplete attention by molecular systematists who focused on subclades with minimal character and/or taxon sampling. As a result, Monarchidae taxonomy is still out-of-date, and biogeographic reconstructions have been based on poorly-resolved phylogenies, limiting their interpretation. Here, we produced a comprehensive, molecular phylogeny of the Monarchidae inferred from mitochondrial and nuclear loci using both concatenated and multilocus coalescent frameworks. We sampled 92% of the 99 recognized monarchid biological species and included deeper sampling within several phylogenetic species complexes, including Monarcha castaneiventris, Symposiachrus barbatus, and Terpsiphone rufiventer. Melampitta is identified as sister to the monarch flycatchers, which themselves comprise four major lineages. The first lineage comprises Terpsiphone and allies, the second lineage is Grallina, the third is Arses and Myiagra, and the fourth lineage comprises a diverse assemblage of genera including the "core monarchs" and the most geographically isolated groups like Chasiempis (Hawaii) and Pomarea (eastern Polynesia). Gene tree discordance was evident in Myiagra, which has implications for basal lineages in the genus (e.g., M. azureocapilla, M. hebetior, and M. alecto). Numerous genera within the core monarchs are paraphyletic, including Mayrornis and Pomarea, whereas the validity of others such as Metabolus are questionable. We recognize polytypic taxa as multiple species, including Lamprolia victoriae and Myiagra azureocapilla. In general, the topology of species complexes included short internodes that were not well resolved, owing to their rapid diversification across island archipelagos. Terpsiphone rufiventer comprises multiple lineages, including a heretofore-unappreciated West African lineage, but relationships within these rapid radiations will require

  18. Lipopolysaccharide Modifications of a Cholera Vaccine Candidate Based on Outer Membrane Vesicles Reduce Endotoxicity and Reveal the Major Protective Antigen

    PubMed Central

    Leitner, Deborah R.; Feichter, Sandra; Schild-Prüfert, Kristina; Rechberger, Gerald N.; Reidl, Joachim

    2013-01-01

    The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay. PMID:23630951

  19. 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. PMID:25054864

  20. Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing

    PubMed Central

    Törnqvist, Elin; Annas, Anita; Granath, Britta; Jalkesten, Elisabeth; Cotgreave, Ian; Öberg, Mattias

    2014-01-01

    The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level. PMID:25054864

  1. 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

  2. Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

    SciTech Connect

    Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

    2012-10-24

    High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

  3. Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks.

    PubMed

    Saini, Camille; Liani, André; Curie, Thomas; Gos, Pascal; Kreppel, Florian; Emmenegger, Yann; Bonacina, Luigi; Wolf, Jean-Pierre; Poget, Yves-Alain; Franken, Paul; Schibler, Ueli

    2013-07-01

    The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase. PMID:23824542

  4. Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks

    PubMed Central

    Saini, Camille; Liani, André; Curie, Thomas; Gos, Pascal; Kreppel, Florian; Emmenegger, Yann; Bonacina, Luigi; Wolf, Jean-Pierre; Poget, Yves-Alain; Franken, Paul; Schibler, Ueli

    2013-01-01

    The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase. PMID:23824542

  5. Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors

    PubMed Central

    Ye, Fan; Breslow, David K; Koslover, Elena F; Spakowitz, Andrew J; Nelson, W James; Nachury, Maxence V

    2013-01-01

    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 PMID:23930224

  6. 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

    2013-01-01

    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. PMID:23930224

  7. Valence-based Word-Face Stroop task reveals differential emotional interference in patients with major depression.

    PubMed

    Başgöze, Zeynep; Gönül, Ali Saffet; Baskak, Bora; Gökçay, Didem

    2015-10-30

    Word-Face Stroop task creates emotional conflict between affective words and affective faces. In this task, healthy participants consistently slow down while responding to incongruent cases. Such interference related slowdown is associated with recruitment of inhibitory processes to eliminate task-irrelevant information. We created a valence-based Word-Face Stroop task, in which participants were asked to indicate whether the words in the foreground are positive, negative or neutral. Healthy participants were faster and more accurate than un-medicated patients with major depression disorder (MDD). In addition, a significant congruence by group interaction is observed: healthy participants slowed down for incongruent cases, but MDD patients did not. Furthermore, for the negative words, healthy individuals made more errors while responding to incongruent cases but MDD patients made the lowest number of errors for this category. The emotional percepts of the patients were intact, because correct response rates in word valence judgments for positive/negative words, and reaction times for happy/sad faces had similar patterns with those of controls. These findings are supported by the analytical rumination interpretation of depression: patients lose speed/accuracy in laboratory tasks due to processing load spent during continuous rumination. However, for tasks in line with their preoccupation, continual practice makes the patients more vigilant and adept. PMID:26272019

  8. Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major

    PubMed Central

    Lamerz, Anne-Christin; Damerow, Sebastian; Kleczka, Barbara; Wiese, Martin; van Zandbergen, Ger; Lamerz, Jens; Wenzel, Alexander; Hsu, Fong-Fu; Turk, John; Beverley, Stephen M.; Routier, Françoise H.

    2010-01-01

    The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:α-d-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis. PMID:20335578

  9. Gender effect on pre-attentive change detection in major depressive disorder patients revealed by auditory MMN.

    PubMed

    Qiao, Zhengxue; Yang, Aiying; Qiu, Xiaohui; Yang, Xiuxian; Zhang, Congpei; Zhu, Xiongzhao; He, Jincai; Wang, Lin; Bai, Bing; Sun, Hailian; Zhao, Lun; Yang, Yanjie

    2015-10-30

    Gender differences in rates of major depressive disorder (MDD) are well established, but gender differences in cognitive function have been little studied. Auditory mismatch negativity (MMN) was used to investigate gender differences in pre-attentive information processing in first episode MDD. In the deviant-standard reverse oddball paradigm, duration auditory MMN was obtained in 30 patients (15 males) and 30 age-/education-matched controls. Over frontal-central areas, mean amplitude of increment MMN (to a 150-ms deviant tone) was smaller in female than male patients; there was no sex difference in decrement MMN (to a 50-ms deviant tone). Neither increment nor decrement MMN differed between female and male patients over temporal areas. Frontal-central MMN and temporal MMN did not differ between male and female controls in any condition. Over frontal-central areas, mean amplitude of increment MMN was smaller in female patients than female controls; there was no difference in decrement MMN. Neither increment nor decrement MMN differed between female patients and female controls over temporal areas. Frontal-central MMN and temporal MMN did not differ between male patients and male controls. Mean amplitude of increment MMN in female patients did not correlate with symptoms, suggesting this sex-specific deficit is a trait- not a state-dependent phenomenon. PMID:26056015

  10. Major mid-late Holocene cooling in the East China Sea revealed by an alkenone sea surface temperature record

    NASA Astrophysics Data System (ADS)

    Zhao, Meixun; Ding, Ling; Xing, Lei; Qiao, Shuqing; Yang, Zuosheng

    2014-12-01

    Although the mid-late Holocene cold and dry event about 4000 years ago (the 4 ka event) has been observed almost globally, it was most prominent in terrestrial climate proxies from the lower latitudes. Here we evaluate the oceanic response to this event in terms of a Holocene sea surface temperature (SST) record reconstructed using the U37K' index for Core B3 on the continental shelf of the East China Sea. The record reveals a large temperature drop of about 5°C from the mid-Holocene (24.7°C at 5.6 ka) to the 4 ka event (19.2°C at 3.8 ka). This mid-late Holocene cooling period in Core B3 correlated with (i) decreases in the East Asia summer monsoon intensity and (ii) the transition period with increased El Niño/Southern Oscillation activities in the Equatorial Pacific. Our SST record provides oceanic evidence for a more global nature of the mid-late Holocene climate change, which was most likely caused by a southward migration of the Intertropical Converge Zone in response to the decreasing summer solar insolation in the Northern Hemisphere. However, the large SST drop around Core B3 indicates that the mid-late Holocene cooling was regionally amplified by the initiation/strengthening of eddy circulation/cold front which caused upwelling and resulted in additional SST decrease. Upwelling during the mid-late Holocene also enhanced with surface productivity in the East China Sea as reflected by higher alkenone content around Core B3.

  11. Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

    PubMed Central

    Dahlhoff, Christoph; Desmarchelier, Charles; Sailer, Manuela; Fürst, Rainer W.; Haag, Alexander; Ulbrich, Susanne E.; Hummel, Björn; Obeid, Rima; Geisel, Jürgen; Bader, Bernhard L.; Daniel, Hannelore

    2013-01-01

    Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine. PMID:23472083

  12. Cyclic Alopecia and Abnormal Epidermal Cornification in Zdhhc13-Deficient Mice Reveal the Importance of Palmitoylation in Hair and Skin Differentiation.

    PubMed

    Liu, Kai-Ming; Chen, Yi-Ju; Shen, Li-Fen; Haddad, Amir N S; Song, I-Wen; Chen, Li-Ying; Chen, Yu-Ju; Wu, Jer-Yuarn; Yen, Jeffrey J Y; Chen, Yuan-Tsong

    2015-11-01

    Many biochemical pathways involved in hair and skin development have not been investigated. Here, we reported on the lesions and investigated the mechanism underlying hair and skin abnormalities in Zdhhc13(skc4) mice with a deficiency in DHHC13, a palmitoyl-acyl transferase encoded by Zdhhc13. Homozygous affected mice showed ragged and dilapidated cuticle of the hair shaft (CUH, a hair anchoring structure), poor hair anchoring ability, and premature hair loss at early telogen phase of the hair cycle, resulting in cyclic alopecia. Furthermore, the homozygous affected mice exhibited hyperproliferation of the epidermis, disturbed cornification, fragile cornified envelope (CE, a skin barrier structure), and impaired skin barrier function. Biochemical investigations revealed that cornifelin, which contains five palmitoylation sites at cysteine residues (C58, C59, C60, C95, and C101), was a specific substrate of DHHC13 and that it was absent in the CUH and CE structures of the affected mice. Furthermore, cornifelin levels were markedly reduced when two palmitoylated cysteines were replaced with serine (C95S and C101S). Taken together, our results suggest that DHHC13 is important for hair anchoring and skin barrier function and that cornifelin deficiency contributes to cyclic alopecia and skin abnormalities in Zdhhc13(skc4) mice. PMID:26121212

  13. 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

  14. Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome

    PubMed Central

    Joyce, Cailin E.; Zhou, Xiang; Xia, Jing; Ryan, Caitriona; Thrash, Breck; Menter, Alan; Zhang, Weixiong; Bowcock, Anne M.

    2011-01-01

    Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 108 small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2–42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin. PMID:21807764

  15. 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

  16. Direct infusion MS-based lipid profiling reveals the pharmacological effects of compound K-reinforced ginsenosides in high-fat diet induced obese mice.

    PubMed

    Shon, Jong Cheol; Shin, Hwa-Soo; Seo, Yong Ki; Yoon, Young-Ran; Shin, Heungsop; Liu, Kwang-Hyeon

    2015-03-25

    The serum lipid metabolites of lean and obese mice fed normal or high-fat diets were analyzed via direct infusion nanoelectrospray-ion trap mass spectrometry followed by multivariate analysis. In addition, lipidomic biomarkers responsible for the pharmacological effects of compound K-reinforced ginsenosides (CK), thus the CK fraction, were evaluated in mice fed high-fat diets. The obese and lean groups were clearly discriminated upon principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) score plot, and the major metabolites contributing to such discrimination were triglycerides (TGs), cholesteryl esters (CEs), phosphatidylcholines (PCs), and lysophosphatidylcholines (LPCs). TGs with high total carbon number (>50) and low total carbon number (<50) were negatively and positively associated with high-fat diet induced obesity in mice, respectively. When the CK fraction was fed to obese mice that consumed a high-fat diet, the levels of certain lipids including LPCs and CEs became similar to those of mice fed a normal diet. Such metabolic markers can be used to better understand obesity and related diseases induced by a hyperlipidic diet. Furthermore, changes in the levels of such metabolites can be employed to assess the risk of obesity and the therapeutic effects of obesity management. PMID:25744175

  17. Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major.

    PubMed

    Crosby, Erika J; Clark, Megan; Novais, Fernanda O; Wherry, E John; Scott, Phillip

    2015-10-01

    Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections. PMID:26290604

  18. Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice.

    PubMed

    Dawson, Paul Anthony; Gardiner, Brooke; Grimmond, Sean; Markovich, Daniel

    2006-07-12

    Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout (Nas1-/-) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1-/- mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased (n = 51) or decreased (n = 41) in Nas1-/- mice when compared with Nas1+/+ mice. The most upregulated transcript levels in Nas1-/- mice were found for the sulfotransferase genes, Sult3a1 (approximately 500% increase) and Sult2a2 (100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes (70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated (> or = 30% increase) in Nas1-/- mice. In addition, Nas1-/- mice exhibited increased levels of hepatic lipid (approximately 16% increase), serum cholesterol (approximately 20% increase), and low-density lipoprotein (approximately 100% increase) and reduced hepatic glycogen (approximately 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1-/- mouse and provide new insights into the metabolic state of the liver in Nas1-/- mice. PMID:16621889

  19. Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.

    PubMed

    Azadi, Mojtaba; Nia, Hadi Tavakoli; Gauci, Stephanie J; Ortiz, Christine; Fosang, Amanda J; Grodzinsky, Alan J

    2016-06-14

    Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid-solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid-solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint

  20. Noninvasive assessment of antenatal hydronephrosis in mice reveals a critical role for Robo2 in maintaining anti-reflux mechanism.

    PubMed

    Wang, Hang; Li, Qinggang; Liu, Juan; Mendelsohn, Cathy; Salant, David J; Lu, Weining

    2011-01-01

    Antenatal hydronephrosis and vesicoureteral reflux (VUR) are common renal tract birth defects. We recently showed that disruption of the Robo2 gene is associated with VUR in humans and antenatal hydronephrosis in knockout mice. However, the natural history, causal relationship and developmental origins of these clinical conditions remain largely unclear. Although the hydronephrosis phenotype in Robo2 knockout mice has been attributed to the coexistence of ureteral reflux and obstruction in the same mice, this hypothesis has not been tested experimentally. Here we used noninvasive high-resolution micro-ultrasonography and pathological analysis to follow the progression of antenatal hydronephrosis in individual Robo2-deficient mice from embryo to adulthood. We found that hydronephrosis progressed continuously after birth with no spontaneous resolution. With the use of a microbubble ultrasound contrast agent and ultrasound-guided percutaneous aspiration, we demonstrated that antenatal hydronephrosis in Robo2-deficient mice is caused by high-grade VUR resulting from a dilated and incompetent ureterovesical junction rather than ureteral obstruction. We further documented Robo2 expression around the developing ureterovesical junction and identified early dilatation of ureteral orifice structures as a potential fetal origin of antenatal hydronephrosis and VUR. Our results thus demonstrate that Robo2 is crucial for the formation of a normal ureteral orifice and for the maintenance of an effective anti-reflux mechanism. This study also establishes a reproducible genetic mouse model of progressive antenatal hydronephrosis and primary high-grade VUR. PMID:21949750

  1. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regions and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.

  2. Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

    PubMed Central

    Ratet, Gwenn; Veyrier, Frédéric J.; Fanton d'Andon, Martine; Kammerscheit, Xavier; Nicola, Marie-Anne; Picardeau, Mathieu; Boneca, Ivo G.; Werts, Catherine

    2014-01-01

    Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to

  3. 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

    2015-08-01

    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. PMID:26045179

  4. Nek8 mutation causes overexpression of galectin-1, sorcin, and vimentin and accumulation of the major urinary protein in renal cysts of jck mice.

    PubMed

    Valkova, Nelly; Yunis, Reem; Mak, Sally K; Kang, Kiandra; Kültz, Dietmar

    2005-07-01

    The jck murine model, which results from a double point mutation in the nek8 gene, has been used to study the mechanism of autosomal recessive polycystic kidney disease (ARPKD). The renal proteome of jck mice was characterized by two-dimensional gel electrophoresis combined with mass spectrometry (MALDI-TOF/TOF). Four newly identified proteins were found to accumulate in the kidneys of jck mice with polycystic kidney disease (PKD) compared with their wild-type littermates. The proteins galectin-1, sorcin, and vimentin were found to be induced 9-, 9-, and 25-fold, respectively, in the PKD proteome relative to the wild type. The identity of these proteins was established by peptide mass fingerprinting and de novo MS/MS sequencing of selected peptides. Up-regulation of these three proteins may be due to the nek8 mutation, and their function may be related to the signaling and structural processes in the primary cilium. Additionally a series of protein isoforms observed only in the ARPKD kidney was identified as the major urinary protein (MUP). Peptide sequencing demonstrated that the isoforms MUP1, MUP2, and MUP6 are contained in this series. The MUP series showed a number of male-specific isoforms and a phosphorylation of the entire series with an increasing degree of phosphorylation of the acidic isoforms. In addition, the MUP series was localized to the cyst fluid of PKD mice, and a cellular mislocalization of galectin-1, sorcin, and vimentin in PKD tubular epithelial cells was shown. The abnormal and extremely high accumulation of the MUPs in the ARPKD kidney may be linked to a defect in protein transport and secretion. The discovery of these proteins will provide new information on the molecular and cellular processes associated with the mechanism of ARPKD. PMID:15872312

  5. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice

    PubMed Central

    Bennewitz, Margaret F.; Watkins, Simon C.; Sundd, Prithu

    2015-01-01

    Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus. PMID:25995970

  6. Interrelationship between lymphocytes and leptin in fat depots of obese mice revealed by changes in nutritional status.

    PubMed

    Lolmède, Karine; Zakaroff-Girard, Alexia; Dray, Cedric; Renoud, Marie-Laure; Daviaud, Danièle; Burcelin, Rémy; Lafontan, Max; Galitzky, Jean; Bouloumié, Anne

    2015-09-01

    The mechanisms underlying the relationships between nutritional status and immunity remain to be fully characterized. The present study was undertaken to analyze by flow cytometry, in the context of diet-induced obesity, the status of immune cells in subcutaneous, and epididymal fat depots in wild-type and immunodeficient Rag2-/- mice submitted to nutritional challenge, i.e., 48-h fasting and 1-week refeeding. In parallel, the responsiveness of mature adipocytes and immune cells in bone marrow, lymph node, and liver were also analyzed. The results show that fasting in obese wild-type mice induces a prominent lipolysis in epididymal AT and immunosuppression restricted to both subcutaneous and epididymal AT, characterized by reduced number of CD4+ T and B lymphocytes and M1/M2 macrophages associated with reduced leptin and increased FGF21 expression in mature adipocytes. One-week refeeding was sufficient to reverse the fasting-induced effects. Obese immunodeficient mice under nutritional challenge exhibited no changes in adipocyte leptin expression and no marked trafficking of AT macrophages or NK cells, while the fasted-induced upregulation of FGF21 expression was maintained as well as the lipolytic responses. The present results demonstrate that, in a context of diet-induced obesity, fasting-induced immunosuppression is restricted to fat depots in immunocompetent mice. Lack of adipocyte leptin regulation and fasting-induced immunosuppression in obese immunodeficient mice strongly suggests that lymphocytes are involved in the modulation of adipocyte leptin expression on one hand and on the other that leptin is involved in the immune changes in AT according to nutritional status. PMID:25670497

  7. 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).

  8. 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

  9. 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. PMID:27528683

  10. Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson’s Disease

    PubMed Central

    Paumier, Katrina L.; Sukoff Rizzo, Stacey J.; Berger, Zdenek; Chen, Yi; Gonzales, Cathleen; Kaftan, Edward; Li, Li; Lotarski, Susan; Monaghan, Michael; Shen, Wei; Stolyar, Polina; Vasilyev, Dmytro; Zaleska, Margaret; D. Hirst, Warren; Dunlop, John

    2013-01-01

    Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments

  11. Behavioral characterization of A53T mice reveals early and late stage deficits related to Parkinson's disease.

    PubMed

    Paumier, Katrina L; Sukoff Rizzo, Stacey J; Berger, Zdenek; Chen, Yi; Gonzales, Cathleen; Kaftan, Edward; Li, Li; Lotarski, Susan; Monaghan, Michael; Shen, Wei; Stolyar, Polina; Vasilyev, Dmytro; Zaleska, Margaret; D Hirst, Warren; Dunlop, John

    2013-01-01

    Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1-2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments

  12. Occurrence of fenestral diaphragms and knots in renal glomerular endothelia of diabetic mutant MafA-/-MafK+ mice as revealed in embedment-free transmission electron microscopy.

    PubMed

    Chaiciwamongkol, Kowit; Toomsan, Yanyong; Arnanteerakul, Tansita; Kondo, Hisatake; Hipkaeo, Wiphawi

    2015-03-01

    Using the advantages (high contrast and transparency and efficient 3D viewing) of embedment-free section transmission electron microscopy (TEM), the occurrence of numerous fenestral diaphragms was clearly shown in 3D en-face viewing of the renal glomerular capillary endothelium of severe overt diabetes mellitus mice, which were generally MafA-deficient and simultaneously MafK-overexpressed specifically in pancreatic β-cells. This presents another example of nephritis-induced diaphragmed fenestrae in the renal glomerular endothelium. In addition, knot-/umbilicus-like structures discrete from and larger than the central knots of regular diaphragms of fenestrated endothelium were clearly demonstrated to occur randomly in the renal glomerular endothelial fenestrae of mutant mice and wild ones. The knot-structures were revealed to be protrusions of underlining basement lamina in conventional TEM by section-tilting observation. PMID:25582093

  13. Automated video analysis system reveals distinct diurnal behaviors in C57BL/6 and C3H/HeN mice.

    PubMed

    Adamah-Biassi, E B; Stepien, I; Hudson, R L; Dubocovich, M L

    2013-04-15

    Advances in rodent behavior dissection using automated video recording and analysis allows detailed phenotyping. This study compared and contrasted 15 diurnal behaviors recorded continuously using an automated behavioral analysis system for a period of 14 days under a 14/10 light/dark cycle in single housed C3H/HeN (C3H) or C57BL/6 (C57) male mice. Diurnal behaviors, recorded with minimal experimental interference and analyzed using phenotypic array and temporal distribution analysis showed bimodal and unimodal profiles in the C57 and C3H mice, respectively. Phenotypic array analysis revealed distinct behavioral rhythms in Activity-Like Behaviors (i.e. walk, hang, jump, come down) (ALB), Exploration-Like Behaviors (i.e. dig, groom, rear up, sniff, stretch) (ELB), Ingestion-Like Behaviors (i.e. drink, eat) (ILB) and Resting-Like Behaviors (i.e. awake, remain low, rest, twitch) (RLB) of C3H and C57 mice. Temporal distribution analysis demonstrated that strain and time of day affects the magnitude and distribution of the spontaneous homecage behaviors. Wheel running activity, water and food measurements correlated with timing of homecage behaviors. Subcutaneous (3 mg/kg, sc) or oral (0.02 mg/ml, oral) melatonin treatments in C57 mice did not modify either the total 24 h magnitude or temporal distribution of homecage behaviors when compared with vehicle treatments. We conclude that C3H and C57 mice show different spontaneous activity and behavioral rhythms specifically during the night period which are not modulated by melatonin. PMID:23337734

  14. Automated Video Analysis System Reveals Distinct Diurnal Behaviors in C57BL/6 and C3H/HeN Mice

    PubMed Central

    Adamah-Biassi, E. B.; Stepien, I.; Hudson, R.L.; Dubocovich, M.L.

    2013-01-01

    Advances in rodent behavior dissection using automated video recording and analysis allows detailed phenotyping. This study compared and contrasted 15 diurnal behaviors recorded continuously using an automated behavioral analysis system for a period of 14 days under a 14/10 light/dark cycle in single housed C3H/HeN (C3H) or C57BL/6 (C57) male mice. Diurnal behaviors, recorded with minimal experimental interference and analyzed using phenotypic array and temporal distribution analysis showed bimodal and unimodal profiles in the C57 and C3H mice, respectively. Phenotypic array analysis revealed distinct behavioral rhythms in activity-like behaviors (i.e. walk, hang, jump, come down) (ALB), exploration-like behaviors (i.e. dig, groom, rear up, sniff, stretch) (ELB), ingestion-like behaviors (i.e. drink, eat) (ILB) and resting-like behaviors (i.e. awake, remain low, rest, twitch) (RLB) of C3H and C57 mice. Temporal analysis demonstrated that strain and time of day affects the magnitude and distribution of the spontaneous homecage behaviors. Wheel running activity, water and food measurements correlated with timing of homecage behaviors. Subcutaneous (3 mg/kg, sc) or oral (0.02 mg/ml, oral) melatonin treatments in C57 mice did not modify either the total 24 hr magnitude or temporal distribution of homecage behaviors when compared with vehicle treatments. We conclude that C3H and C57 mice show different spontaneous activity and behavioral rhythms specifically during the night period which are not modulated by melatonin. PMID:23337734

  15. 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

  16. 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. PMID:25393935

  17. The Unique Functions of the Type-II IL-4 Receptor are revealed in IL-13R¿1-deficient mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The IL-4 receptor is a central mediator of Th2-mediated diseases and associates with either the common gamma chain (type-I IL-4R) or IL-13Ra1 (type-II IL-4R) to form two receptor complexes. Here, using IL-13Ra1-/- mice, we characterized the distinct functions of the type-II IL-4R. In contrast to IL-...

  18. Rescue of Calcineurin Aα−/− Mice Reveals a Novel Role for the α Isoform in the Salivary Gland

    PubMed Central

    Reddy, Ramesh N.; Pena, Juan A.; Roberts, Brian R.; Williams, Stephen R.; Price, S. Russ; Gooch, Jennifer L.

    2011-01-01

    Calcineurin is an important signal transduction mediator in T cells, neurons, the heart, and kidneys. Recent evidence points to unique actions of the two main isoforms of the catalytic subunit. Although the β isoform is required for T-cell development, α is important in the brain and kidney. In addition, mice lacking α but not β suffer from failure to thrive and early mortality. The purpose of this study was to identify the cause of postnatal death of calcineurin α null (CnAα−/−) mice and to determine the mechanism of α activity that contributes to the phenotype. CnAα−/− mice and wild-type littermate controls were fed a modified diet and then salivary gland function and histology were examined. In vitro studies were performed to identify the mechanism of α action. Data show that calcineurin is required for normal submandibular gland function and secretion of digestive enzymes. Loss of α does not impair nuclear factor of activated T-cell activity or expression but results in impaired protein trafficking downstream of the inositol trisphosphate receptor. These findings show a novel function of calcineurin in digestion and protein trafficking. Significantly, these data also provide a mechanism to rescue to adulthood a valuable animal model of calcineurin inhibitor-mediated neuronal and renal toxicities. PMID:21435446

  19. Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1.

    PubMed

    Li, Ji; Jiang, Ting-Xin; Hughes, Michael W; Wu, Ping; Yu, Juehua; Widelitz, Randall B; Fan, Guoping; Chuong, Cheng-Ming

    2012-12-01

    To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14-Cre-mediated loss of DNA methyltransferase 1 (DNMT1). The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to <50% of control in 1-year-old mice. Sections of old mutant skin show follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient-amplifying cells. Although K15-positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further, and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis, which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs. PMID:22763785

  20. Functional interplay between cylindromatosis and histone deacetylase 6 in ciliary homeostasis revealed by phenotypic analysis of double knockout mice.

    PubMed

    Ran, Jie; Yu, Fan; Qin, Juan; Zhang, Yijun; Yang, Yunfan; Li, Dengwen; Zhou, Jun; Liu, Min

    2016-05-10

    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

  1. Incretin Receptor Null Mice Reveal Key Role of GLP-1 but Not GIP in Pancreatic Beta Cell Adaptation to Pregnancy

    PubMed Central

    Moffett, R. Charlotte; Vasu, Srividya; Thorens, Bernard; Drucker, Daniel J.; Flatt, Peter R.

    2014-01-01

    Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1. PMID:24927416

  2. 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

  3. Developmental delay in motor skill acquisition in Niemann-Pick C1 mice reveals abnormal cerebellar morphogenesis.

    PubMed

    Caporali, Paola; Bruno, Francesco; Palladino, Giampiero; Dragotto, Jessica; Petrosini, Laura; Mangia, Franco; Erickson, Robert P; Canterini, Sonia; Fiorenza, Maria Teresa

    2016-01-01

    Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1 (-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1 (nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1 (nmf164) / Npc1 (nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1 (nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-β-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs.These findings indicate that in Npc1 (nmf164) homozygous mice the derangement of synaptic

  4. Genetic regulation analysis reveals involvement of tumor necrosis factor and alpha-induced protein 3 in stress response in mice.

    PubMed

    Xu, Jian; Dai, Aihua; Chen, Qi; Liu, Xiaorong; Zhang, Yu; Wang, Hongmei; Li, Haizhen; Chen, Ying; Cao, Maohong

    2016-01-15

    In order to study whether Tnfaip3 is related to stress response and further to find it's genetic regulation, we use C57BL/6J (B6) and DBA/2 (D2) mice to built the model of chronic unpredictable mild stress. RT-PCR, Western blotting and immunohistochemistry were used for studying the expression variation of Tnfaip3 in hippocampus tissue of B6 and D2 mice after being stressed. We found that the expression of Tnfaip3 was more remarkably increased in chronic unpredictable stress models than that in untreated mice (P<0.05). It is indicated that Tnfaip3 might take part in the process of stress response. The expression of Tnfaip3 is regulated by a cis-acting quantitative trait locus (cis-eQTL). We identified 5 genes are controlled by Tnfaip3 and the expression of 64 genes highly associated with Tnfaip3, 9 of those have formerly been participate in stress related pathways. In order to estimate the relationship between Tnfaip3 and its downstream genes or network members, we transfected SH-SY5Y cells with Tnfaip3 siRNA leading to down-regulation of Tnfaip3 mRNA. We confirmed a significant influence of Tnfaip3 depletion on the expression of Tsc22d3, Pex7, Rap2a, Slc2a3, and Gap43. These validated downstream genes and members of Tnfaip3 gene network provide us new insight into the biological mechanisms of Tnfaip3 in chronic unpredictable stress. PMID:26546835

  5. 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

  6. Sqstm1–GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells

    PubMed Central

    Eino, Atsushi; Kageyama, Shun; Uemura, Takefumi; Annoh, Hiromichi; Saito, Tetsuya; Narita, Ichiei; Waguri, Satoshi; Komatsu, Masaaki

    2015-01-01

    ABSTRACT Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFPKI/+). Using these Sqstm1-GFPKI/+ mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1–GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1–GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFPKI/+ mouse livers caused accumulation of Sqstm1–GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFPKI/+ mice are a useful tool for analyzing Sqstm1 in living cells and intact animals. PMID:26483381

  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

    2014-07-01

    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. PMID:24675475

  8. 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 Central

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

    2014-01-01

    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. PMID:24675475

  9. 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

  10. Analysis of HSD3B7 knockout mice reveals that a 3α-hydroxyl stereochemistry is required for bile acid function

    PubMed Central

    Shea, Heidi C.; Head, Daphne D.; Setchell, Kenneth D. R.; Russell, David W.

    2007-01-01

    Primary bile acids are synthesized from cholesterol in the liver and thereafter are secreted into the bile and small intestine. Gut flora modify primary bile acids to produce secondary bile acids leading to a chemically diverse bile acid pool that is circulated between the small intestine and liver. A majority of primary and secondary bile acids in higher vertebrates have a 3α-hydroxyl group. Here, we characterize a line of knockout mice that cannot epimerize the 3β-hydroxyl group of cholesterol and as a consequence synthesize a bile acid pool in which 3β-hydroxylated bile acids predominate. This alteration causes death in 90% of newborn mice and decreases the absorption of dietary cholesterol in surviving adults. Negative feedback regulation of bile acid synthesis mediated by the farnesoid X receptor (FXR) is disrupted in the mutant mice. We conclude that the correct stereochemistry of a single hydroxyl group at carbon 3 in bile acids is required to maintain their physiologic and regulatory functions in mammals. PMID:17601774

  11. 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. PMID:26289556

  12. Gene-environment interactions reveal a homeostatic role for cholesterol metabolism during dietary folate perturbation in mice

    PubMed Central

    Kitami, Toshimori; Rubio, Renee; O'Brien, William; Quackenbush, John; Nadeau, Joseph H.

    2008-01-01

    Dietary folate supplementation can dramatically reduce the severity and incidence of several common birth defects and adult diseases that are associated with anomalies in homocysteine and folate metabolism. The common polymorphisms that adversely affect these metabolic pathways do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals. To test involvement of folate, homocysteine, and other pathways in disease pathogenesis and treatment response, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice. Compared with the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes. Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and to changes in levels of serum and liver total cholesterol. By genetically increasing serum and liver total cholesterol levels in APOE-deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting that homeostasis among the homocysteine, folate and cholesterol metabolic pathways contributes to the beneficial effects of dietary folate supplementation. PMID:18697859

  13. Optogenetic Stimulation of Arcuate Nucleus Kiss1 Neurons Reveals a Steroid-Dependent Glutamatergic Input to POMC and AgRP Neurons in Male Mice.

    PubMed

    Nestor, Casey C; Qiu, Jian; Padilla, Stephanie L; Zhang, Chunguang; Bosch, Martha A; Fan, Wei; Aicher, Sue A; Palmiter, Richard D; Rønnekleiv, Oline K; Kelly, Martin J

    2016-06-01

    Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, located in the arcuate nucleus (ARC) of the hypothalamus, integrate numerous excitatory and inhibitory inputs to ultimately regulate energy homeostasis. Given that POMC and AgRP neurons are contacted by Kiss1 neurons in the ARC (Kiss1(ARC)) and they express androgen receptors, Kiss1(ARC) neurons may mediate the orexigenic action of testosterone via POMC and/or AgRP neurons. Quantitative PCR analysis of pooled Kiss1(ARC) neurons revealed that mRNA levels for Kiss1 and vesicular glutamate transporter 2 were higher in castrated male mice compared with gonad-intact males. Single-cell RT-PCR analysis of yellow fluorescent protein (YFP) ARC neurons harvested from males injected with AAV1-EF1α-DIO-ChR2:YFP revealed that 100% and 88% expressed mRNAs for Kiss1 and vesicular glutamate transporter 2, respectively. Whole-cell, voltage-clamp recordings from nonfluorescent postsynaptic ARC neurons showed that low frequency photo-stimulation (0.5 Hz) of Kiss1-ChR2:YFP neurons elicited a fast glutamatergic inward current in POMC and AgRP neurons. Paired-pulse, photo-stimulation revealed paired-pulse depression, which is indicative of greater glutamate release, in the castrated male mice compared with gonad-intact male mice. Group I and group II metabotropic glutamate receptor agonists depolarized and hyperpolarized POMC and AgRP neurons, respectively, which was mimicked by high frequency photo-stimulation (20 Hz) of Kiss1(ARC) neurons. Therefore, POMC and AgRP neurons receive direct steroid- and frequency-dependent glutamatergic synaptic input from Kiss1(ARC) neurons in male mice, which may be a critical pathway for Kiss1 neurons to help coordinate energy homeostasis and reproduction. PMID:27093227

  14. Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

    PubMed Central

    Maruyama, Rika; Grevengoed, Elizabeth; Stempniewicz, Peter; Andrew, Deborah J.

    2011-01-01

    Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis. PMID:21698206

  15. 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

  16. Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA.

    PubMed

    Eliaz, Dror; Doniger, Tirza; Tkacz, Itai Dov; Biswas, Viplov Kumar; Gupta, Sachin Kumar; Kolev, Nikolay G; Unger, Ron; Ullu, Elisabetta; Tschudi, Christian; Michaeli, Shulamit

    2015-01-01

    Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications. PMID:25970223

  17. Fitness Assays Reveal Incomplete Functional Redundancy of the HoxA1 and HoxB1 Paralogs of Mice.

    PubMed

    Ruff, James S; Saffarini, Raed B; Ramoz, Leda L; Morrison, Linda C; Baker, Shambralyn; Laverty, Sean M; Tvrdik, Petr; Potts, Wayne K

    2015-10-01

    Gene targeting techniques have led to the phenotypic characterization of numerous genes; however, many genes show minimal to no phenotypic consequences when disrupted, despite many having highly conserved sequences. The standard explanation for these findings is functional redundancy. A competing hypothesis is that these genes have important ecological functions in natural environments that are not needed under laboratory settings. Here we discriminate between these hypotheses by competing mice (Mus musculus) whose Hoxb1 gene has been replaced by Hoxa1, its highly conserved paralog, against matched wild-type controls in seminatural enclosures. This Hoxb1(A1) swap was reported as a genetic manipulation resulting in no discernible embryonic or physiological phenotype under standard laboratory tests. We observed a transient decline in first litter size for Hoxb1(A1) homozygous mice in breeding cages, but their fitness was consistently and more dramatically reduced when competing against controls within seminatural populations. Specifically, males homozygous for the Hoxb1(A1) swap acquired 10.6% fewer territories and the frequency of the Hoxb1(A1) allele decreased from 0.500 in population founders to 0.419 in their offspring. The decrease in Hoxb1(A1) frequency corresponded with a deficiency of both Hoxb1(A1) homozygous and heterozygous offspring. These data suggest that Hoxb1 and Hoxa1 are more phenotypically divergent than previously reported and support that sub- and/or neofunctionalization has occurred in these paralogous genes leading to a divergence of gene function and incomplete redundancy. Furthermore, this study highlights the importance of obtaining fitness measures of mutants in ecologically relevant conditions to better understand gene function and evolution. PMID:26447130

  18. Analysis of Zfhx1a mutant mice reveals palatal shelf contact independent medial edge epithelial differentiation during palate fusion

    PubMed Central

    Jin, Jiu-Zhen; Li, Qun; Higashi, Yujiro; Darling, Douglas S.; Ding, Jixiang

    2008-01-01

    Cleft palate is a common birth defect that involves disruptions in multiple developmental steps such as growth, differentiation, elevation and fusion. Medial edge epithelial (MEE) differentiation is essential for palate fusion. An important question is that the MEE differentiation during fusion is induced by palate shelf contact or is programmed intrinsically by the palate shelf itself. Here, we report that the loss of Zfhx1a function in mice leads to a cleft palate phenotype that is mainly due to a delay in palate elevation. Zfhx1a encodes a transcription regulatory protein that modulates several signaling pathways including those activated by members of the TGF-β superfamily. Loss of Zfhx1a function in mice leads to a complete cleft palate with 100% penetrance. Zfhx1a mutant palatal shelves display normal cell differentiation and proliferation and are able to fuse in an in vitro culture system. The only defect detected was a 24–48 hour delay in palatal shelf elevation. Using the Zfhx1a mutant as a model, we studied the relationship between medial edge epithelial differentiation and palate contact/adhesion. We found that down regulation of Jag2 expression in the medial edge epithelial cells, a key differentiation event establishing palate fusion competence is independent of palate contact/adhesion. Moreover, the expression of several key factors essential for fusion, such as TGF-β3 and MMP13, are also down regulated at stage E16.5 in a contact independent manner, suggesting that differentiation of the medial edge epithelium is largely programmed through an intrinsic mechanism within the palate shelf. PMID:18470539

  19. Touchscreen-paradigm for mice reveals cross-species evidence for an antagonistic relationship of cognitive flexibility and stability.

    PubMed

    Richter, S Helene; Vogel, Anne S; Ueltzhöffer, Kai; Muzzillo, Chiara; Vogt, Miriam A; Lankisch, Katja; Armbruster-Genç, Diana J N; Riva, Marco A; Fiebach, Christian J; Gass, Peter; Vollmayr, Barbara

    2014-01-01

    The abilities to either flexibly adjust behavior according to changing demands (cognitive flexibility) or to maintain it in the face of potential distractors (cognitive stability) are critical for adaptive behavior in many situations. Recently, a novel human paradigm has found individual differences of cognitive flexibility and stability to be related to common prefrontal networks. The aims of the present study were, first, to translate this paradigm from humans to mice and, second, to test conceptual predictions of a computational model of prefrontal working memory mechanisms, the Dual State Theory, which assumes an antagonistic relation between cognitive flexibility and stability. Mice were trained in a touchscreen-paradigm to discriminate visual cues. The task involved "ongoing" and cued "switch" trials. In addition distractor cues were interspersed to test the ability to resist distraction, and an ambiguous condition assessed the spontaneous switching between two possible responses without explicit cues. While response times did not differ substantially between conditions, error rates (ER) increased from the "ongoing" baseline condition to the most complex condition, where subjects were required to switch between two responses in the presence of a distracting cue. Importantly, subjects switching more often spontaneously were found to be more distractible by task irrelevant cues, but also more flexible in situations, where switching was required. These results support a dichotomy of cognitive flexibility and stability as predicted by the Dual State Theory. Furthermore, they replicate critical aspects of the human paradigm, which indicates the translational potential of the testing procedure and supports the use of touchscreen procedures in preclinical animal research. PMID:24834036

  20. 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

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

    PubMed

    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

  2. Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

    PubMed

    Matoba, Yuko; Nonaka, Naoko; Takagi, Yoshitoki; Imamura, Eisaku; Narukawa, Masayuki; Nakamachi, Tomoya; Shioda, Seiji; Banks, William A; Nakamura, Masanori

    2016-09-01

    Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc. PMID:27339371

  3. Unique Mechanisms of Sheng Yu Decoction (聖愈湯 Shèng Yù Tang) on Ischemic Stroke Mice Revealed by an Integrated Neurofunctional and Transcriptome Analysis

    PubMed Central

    Hou, Yu-Chang; Lu, Chung-Kuang; Wang, Yea-Hwey; Chern, Chang-Ming; Liou, Kuo-Tong; Wang, Hsei-Wei; Shen, Yuh-Chiang

    2013-01-01

    Sheng Yu Decoction (聖愈湯 Shèng Yù Tang; SYD) is a popular traditional Chinese medicine (TCM) remedy used in treating cardiovascular and brain-related dysfunction clinically; yet, its neuroprotective mechanisms are still unclear. Here, mice were subjected to an acute ischemic stroke to examine the efficacy and mechanisms of action of SYD by an integrated neurofunctional and transcriptome analysis. More than 80% of the mice died within 2 days after ischemic stroke with vehicle treatment. Treatments with SYD (1.0 g/kg, twice daily, orally or p.o.) and recombinant thrombolytic tissue plasminogen activator (rt-PA; 10 mg/kg, once daily, intravenously or i.v.) both significantly extended the lifespan as compared to that of the vehicle-treated stroke group. SYD successfully restored brain function, ameliorated cerebral infarction and oxidative stress, and significantly improved neurological deficits in mice with stroke. Molecular impact of SYD by a genome-wide transcriptome analysis using brains from stroke mice showed a total of 162 out of 2081 ischemia-induced probe sets were significantly influenced by SYD. Mining the functional modules and genetic networks of these 162 genes revealed a significant upregulation of neuroprotective genes in Wnt receptor signaling pathway (3 genes) and regulation of cell communication (7 genes) and downregulation of destructive genes in response to stress (13 genes) and in the induction of inflammation (5 genes), cytokine production (4 genes), angiogenesis (3 genes), vasculature (6 genes) and blood vessel (5 genes) development, wound healing (7 genes), defense response (7 genes), chemotaxis (4 genes), immune response (7 genes), antigen processing and presenting (3 genes), and leukocyte-mediated cytotoxicity (2 genes) by SYD. Our results suggest that SYD could protect mice against ischemic stroke primarily through significantly downregulating the damaging genes involved in stress, inflammation, angiogenesis, blood vessel formation

  4. 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

  5. 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

    2014-06-01

    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

  6. 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

  7. 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

  8. Whole Exome Sequencing Reveals GUCY2D as a Major Gene Associated With Cone and Cone–Rod Dystrophy in Israel

    PubMed Central

    Lazar, Csilla H.; Mutsuddi, Mousumi; Kimchi, Adva; Zelinger, Lina; Mizrahi-Meissonnier, Liliana; Marks-Ohana, Devorah; Boleda, Alexis; Ratnapriya, Rinki; Sharon, Dror; Swaroop, Anand; Banin, Eyal

    2015-01-01

    Purpose. The Israeli population has a unique genetic make-up, with a high prevalence of consanguineous marriages and autosomal recessive diseases. In rod-dominated phenotypes, disease-causing genes and mutations that differ from those identified in other populations often are incurred. We used whole exome sequencing (WES) to identify genetic defects in Israeli families with cone-dominated retinal phenotypes. Methods. Clinical analysis included family history, detailed ocular examination, visual function testing, and retinal imaging. Whole exome sequencing, followed by segregation analysis, was performed in 6 cone-dominated retinopathy families in which prior mutation analysis did not reveal the causative gene. Based on the WES findings, we screened 106 additional families with cone-dominated phenotypes. Results. The WES analysis revealed mutations in known retinopathy genes in five of the six families: two pathogenic mutations in the GUCY2D gene in three families, and one each in CDHR1 and C8orf37. Targeted screening of additional cone-dominated families led to identification of GUCY2D mutations in four other families, which included two highly probable novel disease-causing variants. Conclusions. Our study suggested that GUCY2D is a major cause of autosomal dominant cone and cone–rod dystrophies in Israel; this is similar to other Caucasian populations and is in contrast with retinitis pigmentosa (primary rod disease), where the genetic make-up of the Israeli population is distinct from other ethnic groups. We also conclude that WES permits more comprehensive and rapid analyses that can be followed by targeted screens of larger samples to delineate the genetic structure of retinal disease in unique population cohorts. PMID:25515582

  9. Slip on faults in the Imperial Valley Triggered by the 4 April 2010 Mw 7.2 El Major earthquake as revealed by InSAR

    NASA Astrophysics Data System (ADS)

    Wei, M.; Sandwell, D. T.; Fialko, Y.; Bilham, R. G.

    2010-12-01

    Radar interferometry (InSAR) reveals surface slip on multiple faults in the Imperial Valley triggered by the 2010 Baja M7.2 earthquake. Co-seismic offsets occurred on the San Andreas, Superstition Hills, Imperial, Elmore Ranch, Wienert, Coyote Creek, Elsinore, Yuha Wells, and several minor faults near the town of Ocotillo at the northern end of the mainshock rupture. We documented right-lateral slip (< 40 mm) on northwest trending faults and left-lateral slip (< 40 mm) on southwest trending faults. Slip occurred on 15-km- and 20-km-long segments of the San Andreas Fault in the Mecca Hills (≤ 50 mm) and Durmid Hill (≤ 10 mm) respectively, and 23 km of the Superstition Hills Fault (≤ 37 mm). Field measurements of slip on the Superstition Hills Fault agree with InSAR and creepmeter measurements to within a few millimeters. Dislocation models of the InSAR data on the Superstition Hills Fault confirm that creep in this sequence, as in previous slip events, is confined to shallow depths (< 3 km). Map of Southern California and Northern Baja California. Black solid lines are major faults. Yellow solid lines are faults with observed offsets. Dashed black lines are National and State borders. White star is the epicenter of the 2010 April 4th El Major earthquake. Red dots are aftershocks within one month after the main rupture with magnitude greater than 5, blue 4-5, and green 3-4. Earthquake data are from the Southern California Earthquake Center. Fault names are abbreviated as follows: ERF, Elmore Ranch fault; SHF, Superstition Hills fault; SMF, Superstition Mountains fault; WF, Wienert fault; YWF, Yuha Wells Fault.

  10. Neurobehavioral phenotyping of Gαq knockout mice reveals impairments in motor functions and spatial working memory without changes in anxiety or behavioral despair

    PubMed Central

    Frederick, Aliya L.; Saborido, Tommy P.; Stanwood, Gregg D.

    2012-01-01

    Many neurotransmitters, hormones, and sensory stimuli elicit their cellular responses through the targeted activation of receptors coupled to the Gαq family of heterotrimeric G proteins. Nevertheless, we still understand little about the consequences of loss of this signaling activity on brain function. We therefore examined the effects of genetic inactivation of Gnaq, the gene that encode for Gαq, on responsiveness in a battery of behavioral tests in order to assess the contribution of Gαq signaling capacity in the brain circuits mediating expression of affective behaviors (anxiety and behavioral despair), spatial working memory, and locomotor output (coordination, strength, spontaneous activity, and drug-induced responses). First, we replicated and extended findings showing clear motor deficits in Gαq knockout mice as assessed on an accelerating rotarod and the inverted screen test. We then assessed the contribution of the basal ganglia motor loops to these impairments, using open field testing and analysis of drug-induced locomotor responses to the psychostimulant cocaine, the benzazepine D1 receptor agonists SKF83822 and SKF83959, and the NMDA receptor antagonist MK-801. We observed significant increases in drug-induced locomotor activity in Gαq knockout mice from the dopaminergic agonists but not MK-801, indicating that basal ganglia locomotor circuitry is largely intact in the absence of Gαq. Additionally, we observed normal phenotypes in both the elevated zero maze and the forced swim test indicating that anxiety and depression-related circuitry appears to be largely intact after loss of Gnaq expression. Lastly, use of the Y-maze revealed spatial memory deficits in Gαq knockout mice, indicating that receptors signaling through Gαq are necessary in these circuits for proficiency in this task. PMID:22723772

  11. Metabonomic Profiling Reveals Cancer Chemopreventive Effects of American Ginseng on Colon Carcinogenesis in Apc(Min/+) Mice.

    PubMed

    Xie, Guoxiang; Wang, Chong-Zhi; Yu, Chunhao; Qiu, Yunping; Wen, Xiao-Dong; Zhang, Chun-Feng; Yuan, Chun-Su; Jia, Wei

    2015-08-01

    American ginseng (Panax quinquefolius L.) is one of the most commonly used herbal medicines in the West. It has been reported to possess significant antitumor effects that inhibit the process of carcinogenesis. However, the mechanisms underlying its anticancer effects remain largely unresolved. In this study, we investigated the cancer chemopreventive effects of American ginseng on the progression of high fat (HF) diet-enhanced colorectal carcinogenesis with a genetically engineered Apc(Min/+) mouse model. The metabolic alterations in sera of experimental mice perturbed by HF diet intervention as well as the American ginseng treatment were measured by gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) analysis. American ginseng treatment significantly extended the life span of the Apc(Min/+) mouse. Significant alterations of metabolites involving amino acids, organic acids, fatty acids, and carbohydrates were observed in Apc(Min/+) mouse in sera, which were attenuated by American ginseng treatment and concurrent with the histopathological improvement with significantly reduced tumor initiation, progression and gut inflammation. These metabolic changes suggest that the preventive effect of American ginseng is associated with attenuation of impaired amino acid, carbohydrates, and lipid metabolism. It also appears that American ginseng induced significant metabolic alterations independent of the Apc(Min/+) induced metabolic changes. The significantly altered metabolites induced by American ginseng intervention include arachidonic acid, linolelaidic acid, glutamate, docosahexaenoate, tryptophan, and fructose, all of which are associated with inflammation and oxidation. This suggests that American ginseng exerts the chemopreventive effects by anti-inflammatory and antioxidant mechanisms. PMID:26136108

  12. BALB/c-congenic ANP32B-deficient mice reveal a modifying locus that determines viability

    PubMed Central

    Leo, Vonny I.; Bunte, Ralph M.; Reilly, Patrick T.

    2015-01-01

    We previously found that deletion of the multifunctional factor ANP32B (a.k.a. SSP29, APRIL, PAL31, PHAPI2) resulted in a severe but strain-specific defect resulting in perinatal lethality. The difficulty in generating an adult cohort of ANP32B-deficient animals limited our ability to examine adult phenotypes, particularly cancer-related phenotypes. We bred the Anp32b-null allele into the BALB/c and FVB/N genetic background. The BALB/c, but not the FVB/N, background provided sufficient frequency of adult Anp32b-null (Anp32b−/−) animals. From these, we found no apparent oncogenic role for this protein in mammary tumorigenesis contrary to what was predicted based on human data. We also found runtism, pathologies in various organ systems, and an unusual clinical chemistry signature in the adult Anp32b−/− mice. Intriguingly, genome-wide single-nucleotide polymorphism analysis suggested that our colony retained an unlinked C57BL/6J locus at high frequency. Breeding this locus to homozygosity demonstrated that it had a strong effect on Anp32b−/− viability indicating that this locus contains a modifier gene of Anp32b with respect to development. This suggests a functionally important genetic interaction with one of a limited number of candidate genes, foremost among them being the variant histone gene H2afv. Using congenic breeding strategies, we have generated a viable ANP32B-deficient animal in a mostly pure background. We have used this animal to reliably exclude mouse ANP32B as an important oncogene in mammary tumorigenesis. Our further phenotyping strengthens the evidence that ANP32B is a widespread regulator of gene expression. These studies may also impact the choice of subsequent groups with respect to congenic breeding versus de novo zygote targeting strategies for background analyses in mouse genetics. PMID:26558540

  13. Metabolomic Profiling of Urine Samples from Mice Exposed to Protons Reveals Radiation Quality and Dose Specific Differences

    PubMed Central

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

    2015-01-01

    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. PMID:25768838

  14. 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

  15. 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. PMID:25768838

  16. Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

    PubMed

    Fenwick, Mark A; Mora, Jocelyn M; Mansour, Yosef T; Baithun, Christina; Franks, Stephen; Hardy, Kate

    2013-09-01

    Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth. PMID:23782946

  17. 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. PMID:21049265

  18. 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

    2016-09-01

    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. PMID:27445329

  19. 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

    2015-01-01

    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 (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. PMID:26434411

  20. 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

  1. 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

  2. Strong evidence from studies with brachymorphic mice and pentachlorophenol that 1'-sulfoöxysafrole is the major ultimate electrophilic and carcinogenic metabolite of 1'-hydroxysafrole in mouse liver.

    PubMed

    Boberg, E W; Miller, E C; Miller, J A; Poland, A; Liem, A

    1983-11-01

    obtained in phenotypically normal B6C3F2 mice of the same age and sex. Brachymorphic mice were also much less responsive than their phenotypically normal littermates to the induction of liver tumors by 1'-hydroxysafrole; lower incidences were observed both when the carcinogen was fed chronically to adult females and when it was administered to males only prior to weaning. Thus, all of these data strongly support the conclusion that 1'-sulfoöxysafrole is the major ultimate electrophilic and tumor-initiating metabolite of 1'-hydroxysafrole. PMID:6577945

  3. 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

  4. T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

    PubMed Central

    Held, W A; Mullins, J J; Kuhn, N J; Gallagher, J F; Gu, G D; Gross, K W

    1989-01-01

    A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2714250

  5. Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia-Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice

    PubMed Central

    Anderson, H. Clarke; Harmey, Dympna; Camacho, Nancy P.; Garimella, Rama; Sipe, Joseph B.; Tague, Sarah; Bi, Xiaohong; Johnson, Kristen; Terkeltaub, Robert; Millán, José Luis

    2005-01-01

    We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2−/−) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1−/− mice is corrected in [Akp2−/−; Enpp1−/−] double-knockout mice. Here we have examined the appendicular skeletons of Akp2−/−, Enpp1−/−, and [Akp2−/−; Enpp1−/−] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2−/−, and [Akp2−/−; Enpp1−/−] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2−/− but also in Enpp1−/− and [Akp2−/−; Enpp1−/−] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1−/− calvarial osteoblasts but decreased mineral deposition by Enpp1−/− long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2−/− mice and the hypomineralized phenotype of the long bones of Enpp1−/− mice are not rescued by simultaneous deletion of TNAP and NPP1 functions. PMID:15920156

  6. 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

  7. 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

  8. Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans

    PubMed Central

    Zampini, Éric; Lepage, Étienne; Tremblay-Belzile, Samuel; Truche, Sébastien; Brisson, Normand

    2015-01-01

    Failure to maintain organelle genome stability has been linked to numerous phenotypes, including variegation and cytosolic male sterility (CMS) in plants, as well as cancer and neurodegenerative diseases in mammals. Here we describe a next-generation sequencing approach that precisely maps and characterizes organelle DNA rearrangements in a single genome-wide experiment. In addition to displaying global portraits of genomic instability, it surprisingly unveiled an abundance of short-range rearrangements in Arabidopsis thaliana and human organelles. Among these, short-range U-turn-like inversions reach 25% of total rearrangements in wild-type Arabidopsis plastids and 60% in human mitochondria. Furthermore, we show that replication stress correlates with the accumulation of this type of rearrangement, suggesting that U-turn-like rearrangements could be the outcome of a replication-dependent mechanism. We also show that U-turn-like rearrangements are mostly generated using microhomologies and are repressed in plastids by Whirly proteins WHY1 and WHY3. A synergistic interaction is also observed between the genes for the plastid DNA recombinase RECA1 and those encoding plastid Whirly proteins, and the triple mutant why1why3reca1 accumulates almost 60 times the WT levels of U-turn-like rearrangements. We thus propose that the process leading to U-turn-like rearrangements may constitute a RecA-independent mechanism to restart stalled forks. Our results reveal that short-range rearrangements, and especially U-turn-like rearrangements, are a major factor of genomic instability in organelles, and this raises the question of whether they could have been underestimated in diseases associated with mitochondrial dysfunction. PMID:25800675

  9. Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

    PubMed Central

    Robledo, Raymond F.; Lambert, Amy J.; Birkenmeier, Connie S.; Cirlan, Marius V.; Cirlan, Andreea Flavia M.; Campagna, Dean R.; Lux, Samuel E.

    2010-01-01

    Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3. PMID:20056793

  10. Early Mechanisms of Pathobiology Are Revealed by Transcriptional Temporal Dynamics in Hippocampal CA1 Neurons of Prion Infected Mice

    PubMed Central

    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

  11. Targeted ablation of the Pde6h gene in mice reveals cross-species differences in cone and rod phototransduction protein isoform inventory.

    PubMed

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C; Huber, Gesine; Seeliger, Mathias W; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-04-17

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3',5'-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h(-/-)) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h(-/-) retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h(-/-) mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  12. 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. PMID:26123489

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

    PubMed Central

    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.

    2014-01-01

    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 TectaL1820F,G1824D/+ mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the TectaC1837G/+ mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the TectaC1619S/+ 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. PMID:24363064

  14. Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory*

    PubMed Central

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C.; Huber, Gesine; Seeliger, Mathias W.; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-01-01

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  15. Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau

    PubMed Central

    Jackson, Samuel J.; Kerridge, Caroline; Cooper, Jane; Cavallini, Annalisa; Falcon, Benjamin; Cella, Claire V.; Landi, Alessia; Szekeres, Philip G.; Murray, Tracey K.; Ahmed, Zeshan; Goedert, Michel; Hutton, Michael; O'Neill, Michael J.

    2016-01-01

    The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. SIGNIFICANCE STATEMENT The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein

  16. Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau.

    PubMed

    Jackson, Samuel J; Kerridge, Caroline; Cooper, Jane; Cavallini, Annalisa; Falcon, Benjamin; Cella, Claire V; Landi, Alessia; Szekeres, Philip G; Murray, Tracey K; Ahmed, Zeshan; Goedert, Michel; Hutton, Michael; O'Neill, Michael J; Bose, Suchira

    2016-01-20

    The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. Significance statement: The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein

  17. 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

  18. Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen.

    PubMed

    Marazuela, Eva G; Rodríguez, Rosalía; Fernández-García, Héctor; García, M Soledad; Villalba, Mayte; Batanero, Eva

    2008-01-01

    Mucosal tolerance induction with vaccines based on peptides representing T-cell epitopes of allergens is a promising way for treating allergic diseases. Ole e 1 is the main allergen of olive pollen, which is an important cause of allergy in Mediterranean countries. The aim of this study was to evaluate the ability of the peptide T109-K130 containing a dominant T-cell epitope of Ole e 1, to modulate the allergen-specific immune response in a prophylactic mouse model. Mice were intranasally treated with the peptide 1 week prior to sensitization with Ole e 1. Blood, lungs and spleens were collected and analysed for immune response. Intranasal pretreatment of mice with the peptide led to suppress serum specific IgE, IgG1 and IgG2a antibody levels, and markedly reduced proliferative T-cell response and Th2-cytokine production, but increased IFN-gamma secretion in spleen cell cultures. Increased mRNA IL-10 levels were observed in lungs from pretreated mice. Pathologic alterations of the lung associated with airway inflammation (peribronchial/perivascular infiltrates, eosinophilia and mucus production) were significantly suppressed after pretreatment. Similar results were obtained when mice were sensitized 10 weeks after treatment. Our results demonstrate that intranasal administration of a single T-cell peptide protects mice against subsequent sensitization to the allergen, possibly via IFN-gamma and IL-10. This study emphasizes the usefulness of nasal peptide T-based vaccines against allergy. PMID:17662454

  19. Mutagenesis Study Reveals the Rim of Catalytic Entry Site of HDAC4 and -5 as the Major Binding Surface of SMRT Corepressor.

    PubMed

    Kim, Gwang Sik; Jung, Ha-Eun; Kim, Jeong-Sun; Lee, Young Chul

    2015-01-01

    Histone deacetylases (HDACs) play a pivotal role in eukaryotic gene expression by modulating the levels of acetylation of chromatin and related transcription factors. In contrast to class I HDACs (HDAC1, -2, -3 and -8), the class IIa HDACs (HDAC4, -5, -7 and -9) harbor cryptic deacetylases activity and recruit the SMRT-HDAC3 complex to repress target genes in vivo. In this regard, the specific interaction between the HDAC domain of class IIa HDACs and the C-terminal region of SMRT repression domain 3 (SRD3c) is known to be critical, but the molecular basis of this interaction has not yet been addressed. Here, we used an extensive mutant screening system, named the "partitioned one- plus two-hybrid system", to isolate SRD3c interaction-defective (SRID) mutants over the entire catalytic domains of HDAC4 (HDAC4c) and -5. The surface presentation of the SRID mutations on the HDAC4c structure revealed that most of the mutations were mapped to the rim surface of the catalytic entry site, strongly suggesting this mutational hot-spot region as the major binding surface of SRD3c. Notably, among the HDAC4c surface residues required for SRD3c binding, some residues (C667, C669, C751, D759, T760 and F871) are present only in class IIa HDACs, providing the molecular basis for the specific interactions between SRD3c and class IIa enzymes. To investigate the functional consequence of SRID mutation, the in vitro HDAC activities of HDAC4 mutants immuno-purified from HEK293 cells were measured. The levels of HDAC activity of the HDAC4c mutants were substantially decreased compared to wild-type. Consistent with this, SRID mutations of HDAC4c prevented the association of HDAC4c with the SMRT-HDAC3 complex in vivo. Our findings may provide structural insight into the binding interface of HDAC4 and -5 with SRD3c, as a novel target to design modulators specific to these enzymes. PMID:26161557

  20. Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species

    PubMed Central

    Rochette, Annie; Raymond, Frédéric; Ubeda, Jean-Michel; Smith, Martin; Messier, Nadine; Boisvert, Sébastien; Rigault, Philippe; Corbeil, Jacques; Ouellette, Marc; Papadopoulou, Barbara

    2008-01-01

    Background Leishmania parasites cause a diverse spectrum of diseases in humans ranging from spontaneously healing skin lesions (e.g., L. major) to life-threatening visceral diseases (e.g., L. infantum). The high conservation in gene content and genome organization between Leishmania major and Leishmania infantum contrasts their distinct pathophysiologies, suggesting that highly regulated hierarchical and temporal changes in gene expression may be involved. Results We used a multispecies DNA oligonucleotide microarray to compare whole-genome expression patterns of promastigote (sandfly vector) and amastigote (mammalian macrophages) developmental stages between L. major and L. infantum. Seven per cent of the total L. infantum genome and 9.3% of the L. major genome were differentially expressed at the RNA level throughout development. The main variations were found in genes involved in metabolism, cellular organization and biogenesis, transport and genes encoding unknown function. Remarkably, this comparative global interspecies analysis demonstrated that only 10–12% of the differentially expressed genes were common to L. major and L. infantum. Differentially expressed genes are randomly distributed across chromosomes further supporting a posttranscriptional control, which is likely to involve a variety of 3'UTR elements. Conclusion This study highlighted substantial differences in gene expression patterns between L. major and L. infantum. These important species-specific differences in stage-regulated gene expression may contribute to the disease tropism that distinguishes L. major from L. infantum. PMID:18510761

  1. Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration.

    PubMed

    Lo, Tsun-Ho; Silveira, Pablo A; Fromm, Phillip D; Verma, Nirupama D; Vu, Phi A; Kupresanin, Fiona; Adam, Rhonda; Kato, Masato; Cogger, Victoria C; Clark, Georgina J; Hart, Derek N J

    2016-08-01

    C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration. PMID:27316686

  2. 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. PMID:27443888

  3. 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

  4. Fibroblast Growth Factor 2 Internal Ribosome Entry Site (Ires) Activity Ex Vivo and in Transgenic Mice Reveals a Stringent Tissue-Specific Regulation

    PubMed Central

    Créancier, Laurent; Morello, Dominique; Mercier, Pascale; Prats, Anne-Catherine

    2000-01-01

    Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 −/− osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system. PMID:10893274

  5. Likelihood and Bayesian analyses reveal major genes affecting body composition, carcass, meat quality and the number of false teats in a Chinese European pig line

    PubMed Central

    Marie-Pierre, Sanchez; Bidanel, Jean-Pierre; Zhang, Siqing; Naveau, Jean; Burlot, Thierry; Le Roy, Pascale

    2003-01-01

    Segregation analyses were performed using both maximum likelihood – via a Quasi Newton algorithm – (ML-QN) and Bayesian – via Gibbs sampling – (Bayesian-GS) approaches in the Chinese European Tiameslan pig line. Major genes were searched for average ultrasonic backfat thickness (ABT), carcass fat (X2 and X4) and lean (X5) depths, days from 20 to 100 kg (D20100), Napole technological yield (NTY), number of false (FTN) and good (GTN) teats, as well as total teat number (TTN). The discrete nature of FTN was additionally considered using a threshold model under ML methodology. The results obtained with both methods consistently suggested the presence of major genes affecting ABT, X2, NTY, GTN and FTN. Major genes were also suggested for X4 and X5 using ML-QN, but not the Bayesian-GS, approach. The major gene affecting FTN was confirmed using the threshold model. Genetic correlations as well as gene effect and genotype frequency estimates suggested the presence of four different major genes. The first gene would affect fatness traits (ABT, X2 and X4), the second one a leanness trait (X5), the third one NTY and the last one GTN and FTN. Genotype frequencies of breeding animals and their evolution over time were consistent with the selection performed in the Tiameslan line. PMID:12927073

  6. 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.

  7. 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…

  8. BALB/c and SWR inbred mice differ in post-oral fructose appetition as revealed by sugar versus non-nutritive sweetener tests.

    PubMed

    Kraft, Tamar T; Huang, Donald; Lolier, Melanie; Warshaw, Deena; LaMagna, Sam; Natanova, Elona; Sclafani, Anthony; Bodnar, Richard J

    2016-01-01

    Recent studies indicate that C57BL/6J (B6) and FVB inbred mouse strains differ in post-oral fructose conditioning. This was demonstrated by their differential flavor conditioning response to intragastric fructose and their preference for fructose versus a non-nutritive sweetener. The present study extended this analysis to SWR and BALB/c inbred strains which are of interest because they both show robust flavor conditioning responses to fructose. In the first experiment, ad-libitum fed mice were given a series of 2-day, two-bottle preference tests between 8% fructose and a more preferred, but non-nutritive 0.1% sucralose +0.1% saccharin (S+S) solution (tests 1 & 4), and fructose or S+S versus water (tests 2 and 3). In test 1, SWR mice preferred S+S to fructose, and in tests 2 and 3, they preferred both sweeteners to water. In test 4, SWR mice switched their preference and consumed more fructose than S+S. In contrast, ad-libitum fed BALB/c mice strongly preferred S+S to fructose in both tests 1 and 4, although they preferred both sweeteners to water in tests 2 and 3. Food-restricted BALB/c mice also preferred the non-nutritive S+S to fructose in tests 1 and 4. The experience-induced fructose preference reversal observed in SWR, but not BALB/c mice indicates that fructose has a post-oral reinforcing effect in SWR mice as in FVB mice. Because B6 and FVB mice prefer glucose to fructose based on the post-oral actions of the two sugars, the second experiment compared the preferences of SWR and BALB/c mice for 8% glucose and fructose solutions. Ad-libitum fed and food-restricted SWR mice strongly preferred glucose to fructose. In contrast, ad-libitum fed BALB/c mice were indifferent to the sugars, perhaps because of their overall low intakes. Food-restricted BALB/c mice, however, strongly preferred glucose. These findings indicate that SWR and BALB/c mice differ in their preference response to the post-oral actions of fructose. PMID:26485292

  9. 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

  10. Genomic Profiling of Collaborative Cross Founder Mice Infected with Respiratory Viruses Reveals Novel Transcripts and Infection-Related Strain-Specific Gene and Isoform Expression

    PubMed Central

    Xiong, Hao; Morrison, Juliet; Ferris, Martin T.; Gralinski, Lisa E.; Whitmore, Alan C.; Green, Richard; Thomas, Matthew J.; Tisoncik-Go, Jennifer; Schroth, Gary P.; Pardo-Manuel de Villena, Fernando; Baric, Ralph S.; Heise, Mark T.; Peng, Xinxia; Katze, Michael G.

    2014-01-01

    Genetic variation between diverse mouse species is well-characterized, yet existing knowledge of the mouse transcriptome comes largely from one mouse strain (C57BL/6J). As such, it is unlikely to reflect the transcriptional complexity of the mouse species. Gene transcription is dynamic and condition-specific; therefore, to better understand the mouse transcriptional response to respiratory virus infection, we infected the eight founder strains of the Collaborative Cross with either influenza A virus or severe acute respiratory syndrome coronavirus and sequenced lung RNA samples at 2 and 4 days after infection. We found numerous instances of transcripts that were not present in the C57BL/6J reference annotation, indicating that a nontrivial proportion of the mouse genome is transcribed but poorly annotated. Of these novel transcripts, 2150 could be aligned to human or rat genomes, but not to existing mouse genomes, suggesting functionally conserved sequences not yet recorded in mouse genomes. We also found that respiratory virus infection induced differential expression of 4287 splicing junctions, resulting in strain-specific isoform expression. Of these, 59 were influenced by strain-specific mutations within 2 base pairs of key intron–exon boundaries, suggesting cis-regulated expression. Our results reveal the complexity of the transcriptional response to viral infection, previously undocumented genomic elements, and extensive diversity in the response across mouse strains. These findings identify hitherto unexplored transcriptional patterns and undocumented transcripts in genetically diverse mice. Host genetic variation drives the complexity and diversity of the host response by eliciting starkly different transcriptional profiles in response to a viral infection. PMID:24902603

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

    PubMed

    Beauchemin, Kyle J; Wells, Julie M; Kho, Alvin T; Philip, Vivek M; Kamir, Daniela; Kohane, Isaac S; Graber, Joel H; Bult, Carol J

    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

  12. 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

  13. Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

    PubMed Central

    Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

    2014-01-01

    Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

  14. Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

    PubMed

    Sukup-Jackson, Michelle R; Kiraly, Orsolya; Kay, Jennifer E; Na, Li; Rowland, Elizabeth A; Winther, Kelly E; Chow, Danielle N; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S; Maklakova, Vilena I; Singh, Vijay R; Wadduwage, Dushan N; Rajapakse, Jagath; So, Peter T C; Collier, Lara S; Engelward, Bevin P

    2014-06-01

    Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

  15. Leishmania major: differential resistance to infection in C57BL/6 (high interferon-alpha/beta) and congenic B6.C-H-28c (low interferon-alpha/beta) mice.

    PubMed

    Shankar, A H; Morin, P; Titus, R G

    1996-11-01

    In murine cutaneous leishmaniasis caused by Leishmania major (Lm), resistance often associates with the outgrowth of Lm-specific Th1 cells. Parasites are eliminated by Th1-mediated activation of infected macrophages (M phi) which destroy Lm by producing toxic nitrogen and oxygen radicals. The cytokine IFN-alpha activates microbicidal functions of M phis and facilitates outgrowth of Th1 cells. Therefore, we compared the course of infection with Lm in resistant C57BL/6 mice, bearing the If-1h high expression allele for IFN-alpha/beta, with the congenic B6.C-H-28c mouse, bearing the If-1I low expression allele from the Lm-susceptible BALB/c strain. We observed that B6.C-H-28c animals developed up to 70% larger footpad lesions and harbored up to 1000-fold more parasites than C57BL/6 mice. Furthermore, peak Lm-specific IFN-gamma production in the B6.C-H-28c animals was lower and delayed by approximately 2 weeks, whereas IL-4 production was higher and persisted approximately 2 weeks longer. Since these results suggested that IFN-alpha/beta plays a protective role in mice infected with Lm, we determined whether infusing B6.C-H-28c mice with IFN-alpha would influence the course of infection with Lm. Unfortunately, the mice developed severe peritoneal hemorrhaging in response to injection with IFN-alpha. Therefore, we examined the ability of IFN-alpha to activate M phis to destroy Lm in vitro. We observed that rIFN-alpha could synergize with subactivating doses of LPS to activate both C57BL/6 and BALB/c peritoneal M phis to produce NO and to kill intracellular Lm. Taken as a whole, these results suggest that type I interferons may play a protective role in cutaneous leishmaniasis. PMID:8932763

  16. 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

  17. Disposition of the Herbicide 2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine) and Its Major Metabolites in Mice: A Liquid Chromatography/Mass Spectrometry Analysis of Urine, Plasma, and Tissue Levels

    PubMed Central

    Ross, Matthew K.; Jones, Toni L.; Filipov, Nikolay M.

    2009-01-01

    2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine, ATR) is a toxicologically important and widely used herbicide. Recent studies have shown that it can elicit neurological, immunological, developmental, and biochemical alterations in several model organisms, including in mice. Because disposition data in mice are lacking, we evaluated ATR's metabolism and tissue dosimetry after single oral exposures (5–250 mg/kg) in C57BL/6 mice using liquid chromatography/mass spectrometry (Ross and Filipov, 2006). ATR was metabolized and cleared rapidly; didealkyl ATR (DACT) was the major metabolite detected in urine, plasma, and tissues. Plasma ATR peaked at 1 h postdosing and rapidly declined, whereas DACT peaked at 2 h and slowly declined. Most ATR and metabolite residues were excreted within the first 24 h. However, substantial amounts of DACT were still present in 25- to 48-h and 49- to 72-h urine. ATR reached maximal brain levels (0.06–1.5 μM) at 4 h (5–125 mg/kg) and 1 h (250 mg/kg) after dosing, but levels quickly declined to <0.1 μM by 12 h in all the groups. In contrast, strikingly high concentrations of DACT (1.5–50 μM), which are comparable with liver DACT levels, were detectable in brain at 2 h. Brain DACT levels slowly declined, paralleling the kinetics of plasma DACT. Our findings suggest that in mice ATR is widely distributed and extensively metabolized and that DACT is a major metabolite detected in the brain at high levels and is ultimately excreted in urine. Our study provides a starting point for the establishment of models that link target tissue dose to biological effects caused by ATR and its in vivo metabolites. PMID:19116264

  18. 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. PMID:25284342

  19. Bone marrow chimeras and c-fms conditional ablation (Mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation.

    PubMed

    Chinnery, Holly R; Carlson, Eric C; Sun, Yan; Lin, Michelle; Burnett, Sandra H; Perez, Victor L; McMenamin, Paul G; Pearlman, Eric

    2009-03-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4(-/-) mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)(+)/C57BL/6 or eGFP(+)/TLR4(-/-) mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4(-/-) mice reconstituted with C57BL/6, but not TLR4(-/-) bone marrow cells donor cells were found to cause infiltration of eGFP(+) cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP(+) cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1alpha production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation. PMID:19234168

  20. Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation1

    PubMed Central

    Chinnery, Holly R.; Carlson, Eric C.; Sun, Yan; Lin, Michelle; Burnett, Sandra H.; Perez, Victor L.; McMenamin, Paul G.; Pearlman, Eric

    2012-01-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation. PMID:19234168

  1. A QTL study on late leaf spot and rust revealed one major QTL for molecular breeding for rust resistance in groundnut (Arachis hypogaea L.)

    PubMed Central

    Khedikar, Y. P.; Gowda, M. V. C.; Sarvamangala, C.; Patgar, K. V.; Upadhyaya, H. D.

    2010-01-01

    Late leaf spot (LLS) and rust are two major foliar diseases of groundnut (Arachis hypogaea L.) that often occur together leading to 50–70% yield loss in the crop. A total of 268 recombinant inbred lines of a mapping population TAG 24 × GPBD 4 segregating for LLS and rust were used to undertake quantitative trait locus (QTL) analysis. Phenotyping of the population was carried out under artificial disease epiphytotics. Positive correlations between different stages, high to very high heritability and independent nature of inheritance between both the diseases were observed. Parental genotypes were screened with 1,089 simple sequence repeat (SSR) markers, of which 67 (6.15%) were found polymorphic. Segregation data obtained for these markers facilitated development of partial linkage map (14 linkage groups) with 56 SSR loci. Composite interval mapping (CIM) undertaken on genotyping and phenotyping data yielded 11 QTLs for LLS (explaining 1.70–6.50% phenotypic variation) in three environments and 12 QTLs for rust (explaining 1.70–55.20% phenotypic variation). Interestingly a major QTL associated with rust (QTLrust01), contributing 6.90–55.20% variation, was identified by both CIM and single marker analysis (SMA). A candidate SSR marker (IPAHM 103) linked with this QTL was validated using a wide range of resistant/susceptible breeding lines as well as progeny lines of another mapping population (TG 26 × GPBD 4). Therefore, this marker should be useful for introgressing the major QTL for rust in desired lines/varieties of groundnut through marker-assisted backcrossing. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1366-x) contains supplementary material, which is available to authorized users. PMID:20526757

  2. 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

  3. RNA sequencing reveals high resolution expression change of major plant hormone pathway genes after young seedless grape berries treated with gibberellin.

    PubMed

    Chai, Lijuan; Li, Yanmei; Chen, Shangwu; Perl, Avihai; Zhao, Fengxia; Ma, Huiqin

    2014-12-01

    Seedless varieties are of particular importance to the table-grape and raisin industries. Gibberellin (GA) application is widely used in the early stages of seedless berry development to increase berry size and economic value. However, the underlying mechanism of GA induction of berry enlargement is not well understood. Here, RNA-sequencing analysis of 'Centennial Seedless' (Vitis vinifera L.) berries treated with GA3 12 days after flowering is reported. Pair-wise comparison of GA3-treated and control samples detected 165, 444, 463 genes with an over two-fold change in expression 1, 3, and 7 days after GA3 treatment, respectively. The number of differentially expressed genes increased with time after GA3 treatment, and the differential expression was dominated by downregulation. Significantly modulated expression included genes encoding synthesis and catabolism to manage plant hormone homeostasis, hormone transporters, receptors and key components in signaling pathways; exogenous GA3 induced multipoint cross talk with auxin, cytokinin, brassinosteroid, ABA and ethylene. The temporal gene-expression patterns of cell-wall-modification enzymes, cytoskeleton and membrane components and transporters revealed a pivotal role for cell-wall-relaxation genes in GA3-induced berry enlargement. Our results provide the first sequential transcriptomic atlas of exogenous GA3-induced berry enlargement and reveal the complexity of GA3's effect on berry sizing. PMID:25443848

  4. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans.

    PubMed

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G; Van Etten, James L

    2013-08-20

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

  5. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

    PubMed Central

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

    2013-01-01

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

  6. Expression of pancreatic and duodenal homeobox1 (PDX1) protein in the interior and exterior regions of the intestine, revealed by development and analysis of Pdx1 knockout mice

    PubMed Central

    Kamisako, Tsutomu; Kagawa, Takahiro; Haraguchi, Seiki; Yagoto, Mika; Takahashi, Ri-ichi; Kawai, Kenji; Suemizu, Hiroshi

    2015-01-01

    We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the β cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129+Ter/SvJcl×C57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1-/-) mice. PMID:26155204

  7. The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice

    PubMed Central

    Stewart, Michael K. G.; Gong, Xiang-Qun; Barr, Kevin J.; Bai, Donglin; Fishman, Glenn I.; Laird, Dale W.

    2012-01-01

    Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients. PMID:23075222

  8. 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

  9. Systematic Definition of Protein Constituents along the Major Polarization Axis Reveals an Adaptive Reuse of the Polarization Machinery in Pheromone-Treated Budding Yeast

    PubMed Central

    2008-01-01

    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. PMID:19053807

  10. DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice.

    PubMed

    Ahmed, Sami Ben Hadj; Touihri, Leila; Chtourou, Yessine; Dellagi, Koussay; Bahloul, Chokri

    2009-01-01

    Despite the lack of effective vaccines against parasitic diseases, the prospects of developing a vaccine against leishmaniasis are still high. With this objective, we have tested four DNA based candidate vaccines encoding to immunodominant leishmania antigens (LACKp24, TSA, LmSTI1 and CPa). These candidates have been previously reported as capable of eliciting at least partial protections in the BALB/c mice model of experimental cutaneous leishmaniasis. When tested under similar experimental conditions, all of them were able to induce similar partial protective effects, but none could induce a full protection. In order to improve the level of protection we have explored the approach of DNA based vaccination with different cocktails of plasmids encoding to the different immunodominant Leishmania antigens. A substantial increase of protection was achieved when the cocktail is composed of all of the four antigens; however, no full protection was achieved when mice were challenged with a high dose of parasite in their hind footpad. The full protection was only achieved after a challenge with a low parasitic dose in the dermis of the ear. It was difficult to determine clear protection correlates, other than the mixture of immunogens induced specific Th1 immune responses against each component. Therefore, such an association of antigens increased the number of targeted epitopes by the immune system with the prospects that the responses are at least additive if not synergistic. Even though, any extrapolation of this approach when applied to other animal or human models is rather hazardous, it undoubtedly increases the hopes of developing an effective leishmania vaccine. PMID:18951941

  11. Conditional knockout of the leptin receptor in the colonic epithelium revealed the local effects of leptin receptor signaling in the progression of colonic tumors in mice.

    PubMed

    Higurashi, Takuma; Endo, Hiroki; Uchiyama, Takashi; Uchiyama, Shiori; Yamada, Eiji; Ohkubo, Hidenori; Sakai, Eiji; Takahashi, Hirokazu; Maeda, Shin; Wada, Koichiro; Natsumeda, Yutaka; Hippo, Yoshitaka; Nakajima, Atsushi; Nakagama, Hitoshi

    2014-09-01

    Leptin, secreted by the adipose tissue and known to be related to obesity, is considered to be involved in the onset and progression of colorectal cancer. However, the exact role of leptin in colorectal carcinogenesis is still unclear, as several controversial reports have been published on the various systemic effects of leptin. The aim of this study was to clarify the local and precise roles of leptin receptor (LEPR)-mediated signaling in colonic carcinogenesis using intestinal epithelium-specific LEPRb conditional knockout (cKO) mice. We produced and used colonic epithelium-specific LEPRb cKO mice to investigate the carcinogen-induced formation of aberrant crypt foci (ACF) and tumors in the colon, using their littermates as control. There were no differences in the body weight or systemic condition between the control and cKO mice. The tumor sizes and number of large-sized tumors were significantly lower in the cKO mice as compared with those in the control mice. On the other hand, there was no significant difference in the proliferative activity of the normal colonic epithelial cells or ACF formation between the control and cKO mice. In the control mice, marked increase of the LEPRb expression level was observed in the colonic tumors as compared with that in the normal epithelium; furthermore, signal transducer and activator of transcription (STAT3) was activated in the tumor cells. These findings suggest that STAT3 is one of the important molecules downstream of LEPRb, and LEPRb/STAT3 signaling controls tumor cell proliferation. We demonstrated the importance of local/regional LEPR-mediated signaling in colorectal carcinogenesis. PMID:24958593

  12. The Shallow Plumbing System of Piton de la Fournaise Volcano (La Réunion island, Indian Ocean) Revealed by the Major 2007 Caldera Forming Eruption (Invited)

    NASA Astrophysics Data System (ADS)

    Di Muro, A.; Metrich, N.; Daniele, V.; Rosi, M.; Armienti, P.; Fougeroux, T.; Deloule, E.; Arienzo, I.; Civetta, L.

    2013-12-01

    The 2007 eruption represents a major event in the recent history of Piton de la Fournaise volcano because it produced: i) the most voluminous lava field (at least 0.21 km3), ii) the most intense lava fountaining activity (>200 m high), iii) the largest SO2 plume (>230 kt), iv) the largest summit collapse (1 km wide x 0.34 km deep) and v) the main flank slip event (up to 1.4 m eastwards) ever documented at PdF. The bulk magma volume extruded during the 2007 eruptive sequence is similar to that emitted during the entire 1998-2006 period. As a whole, the volume of magma emitted during the whole 1998-2007 activity cycle is remarkably close to that estimated (~0.35 km3) for the shallow plumbing system of Piton de la Fournaise. The 2007 eruptive sequence consisted of three successive phases (February, March and April). The main caldera forming phase of April ended a 9 years long period (1998-2007) of continuous edifice inflation and frequent eruptive activity (3 eruptions per year on average). On the contrary, post-2007 activity punctuates a trend of continuous deflation and consists of small-volume summit eruptions of moderate/low MgO magmas and frequent shallow magma intrusions. The 2007 lavas and pyroclasts cover the complete range of textures and crystal content an composition typically found in PdF products. The broad range of textures and the large volumes of pyroclasts, lava and gas emitted in 2007 provide an unique and exceptional record of the time-integrated evolution of PdF magma, and represent an unique opportunity to image the volcano plumbing system and bring new constraints on the processes controlling its magmatic and volatile budget. We here address these issues by using an unprecedented geochemical dataset (major, volatile and trace elements, Sr-Nd isotopes) on bulk rocks, minerals, glass inclusions and glass matrices from a very detailed sample set, representative of the time evolution of extruded magma during the entire 2007 eruptive sequence.

  13. Minor influence of lifelong voluntary exercise on composition, structure, and incidence of osteoarthritis in tibial articular cartilage of mice compared with major effects caused by growth, maturation, and aging.

    PubMed

    Närhi, Tommi; Siitonen, Ulrika; Lehto, Lauri J; Hyttinen, Mika M; Arokoski, Jari P A; Brama, Pieter A; Jurvelin, Jukka S; Helminen, Heikki J; Julkunen, Petro

    2011-10-01

    We investigated the effects of lifelong voluntary exercise on articular cartilage of mice. At the age of 4 weeks C57BL mice (n = 152) were divided into two groups, with one group serving as a sedentary control whereas the other was allowed free access to a running wheel from the age of 1 month onward. Mice were euthanized at four different time points (1, 2, 6, and 18 months of age). Articular cartilage samples were gathered from the load-bearing area of the tibial medial plateaus, and osteoarthritis was graded. Additionally, the proteoglycan content distribution was assessed using digital densitometry, collagen fibril orientation, and parallelism with polarized light microscopy, and collagen content using Fourier transform infrared imaging spectroscopy. The incidence of osteoarthritis increased with aging, but exercise had no effect on this trend. Furthermore, the structure and composition revealed significant growth, maturation, and age-dependent properties. Exercise exerted a minor effect on collagen fibril orientation in the superficial zone. Fibril orientation at 2 months of age was more perpendicular to surface (p < 0.05) in controls compared with runners, whereas the situation was reversed at the age of 18 months (p < 0.05). The collagen content of the superficial zone was higher (p < 0.01) at the age of 18 months in controls compared with runners but the proteoglycan content did not display any exercise-dependent changes. In conclusion, growth, maturation, and aging exerted a clear effect on integrity, structure, and composition of medial tibial plateau articular cartilage in mice, whereas lifelong voluntary exercise had only a minor effect on collagen architecture and content. PMID:21405978

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

    PubMed

    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

    2009-11-01

    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 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 type III secretion system 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

  15. 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

  16. Multigene phylogenetic reconstruction of the Tubulinea (Amoebozoa) corroborates four of the six major lineages, while additionally revealing that shell composition does not predict phylogeny in the Arcellinida.

    PubMed

    Lahr, Daniel J G; Grant, Jessica R; Katz, Laura A

    2013-05-01

    Tubulinea is a phylogenetically stable higher-level taxon within Amoebozoa, morphologically characterized by monoaxially streaming and cylindrical pseudopods. Contemporary phylogenetic reconstructions have largely relied on SSU rDNA, and to a lesser extent, on actin genes to reveal the relationships among these organisms. Additionally, the test (shell) forming Arcellinida, one of the most species-rich amoebozoan groups, is nested within Tubulinea and suffers from substantial under-sampling of taxa. Here, we increase taxonomic and gene sampling within the Tubulinea, characterizing molecular data for 22 taxa and six genes (SSU rDNA, actin, α- and β-tubulin, elongation factor 2 and the 14-3-3 regulatory protein). We perform concatenated phylogenetic analyses using these genes as well as approximately unbiased tests to assess evolutionary relationships within the Tubulinea. We confirm the monophyly of Tubulinea and four of the six included lineages (Echinamoeboidea, Leptomyxida, Amoebida and Poseidonida). Arcellinida and Hartmanellidae, the remaining lineages, are not monophyletic in our reconstructions, although statistical testing does not allow rejection of either group. We further investigate more fine-grained morphological evolution of previously defined groups, concluding that relationships within Arcellinida are more consistent with general test and aperture shape than with test composition. We also discuss the implications of this phylogeny for interpretations of the Precambrian fossil record of testate amoebae. PMID:23499265

  17. 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

  18. Transcriptome Profiling, Molecular Biological, and Physiological Studies Reveal a Major Role for Ethylene in Cotton Fiber Cell Elongation[W][OA

    PubMed Central

    Shi, Yong-Hui; Zhu, Sheng-Wei; Mao, Xi-Zeng; Feng, Jian-Xun; Qin, Yong-Mei; Zhang, Liang; Cheng, Jing; Wei, Li-Ping; Wang, Zhi-Yong; Zhu, Yu-Xian

    2006-01-01

    Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes. PMID:16461577

  19. Transcriptome analysis reveals an activation of major histocompatibility complex 1 and 2 pathways in chicken trachea immunized with infectious laryngotracheitis virus vaccine.

    PubMed

    Luo, Juan; Carrillo, José A; Menendez, Kimberly R; Tablante, Nathaniel L; Song, Jiuzhou

    2014-04-01

    Infectious laryngotracheitis is an acute, contagious, upper respiratory disease of chickens caused by gallid herpes virus 1. Due to mortality rates that can reach up to 70% depending on the virulence of the virus, the disease is of great economic importance to the poultry industry. In this study, 15-d-old specific pathogen-free White Leghorn chickens were used to perform transcriptome analysis of chicken trachea immunized with infectious laryngotracheitis virus vaccine. Myosin and several collagen-related genes were downregulated in the immunized group, suggesting that normal function and structure may be compromised. In addition, we identified some cytokine receptors and several immune genes, such as Granzyme A (GZMA), CD4 molecule (CD4), CD8a molecule (CD8A), and CD8b molecule (CD8B), that were upregulated upon vaccination. The gene ontology analysis shows that genes included in the biological process cluster were related to antigen processing and presentation, positive regulation of immune system processes, T cell selection, and positive regulation of T cell activation. In conclusion, chicken embryo origin vaccine activation of the major histocompatibility complex 1 and 2 pathways provides insight for evaluation and design of infectious laryngotracheitis vaccines. PMID:24706961

  20. Petrogenesis of tholeiitic basalts from the Central Atlantic magmatic province as revealed by mineral major and trace elements and Sr isotopes

    NASA Astrophysics Data System (ADS)

    Marzoli, Andrea; Jourdan, Fred; Bussy, François; Chiaradia, Massimo; Costa, Fidel

    2014-02-01

    The petrogenesis of the Kerforne dyke tholeiitic basalts (Brittany, France), the northernmost outcrop of the 200 Ma Central Atlantic magmatic province (CAMP), is constrained by its zoned augite and plagioclase crystals. Augite cores with high Mg/Fe and Cr suggest crystallization from near-primary magmas, with slightly enriched Rare Earth element (REE) patterns. Plagioclase crystals with high-An (An85) rounded cores are MgO- and K-rich, REE-poor, and display 87Sr/86Sr200Ma (0.7058) significantly higher than those of the surrounding ground-mass (0.7052-0.7053) suggesting open-system evolution processes. We propose a differentiation process involving mixing of different mafic magmas which occurred in less than a few hundred years judging from the lack of diffusive re-equilibration of major and trace elements in augite and of 87Sr/86Sr200Ma in plagioclase cores. The relatively large range of incompatible element contents and ratios of observed and calculated magmas are possibly due to fractional crystallization and to moderate amounts of crustal contamination which affected the more primitive magmas in particular. The calculated magmas reach near-primitive compositions and suggest that they originated from melting of a spinel peridotite slightly enriched in LREE vs. HREE.

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

    PubMed

    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

  2. Subpopulation-proteomics reveal growth rate, but not cell cycling, as a major impact on protein composition in Pseudomonas putida KT2440.

    PubMed

    Lieder, Sarah; Jahn, Michael; Seifert, Jana; von Bergen, Martin; Müller, Susann; Takors, Ralf

    2014-01-01

    Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery. In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells. PMID:25401072

  3. 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.

  4. 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 an unusual cell envelope that includes a loose-fitting sheath around each cell, often called a toga. Only two toga-associated structural proteins have been identified in Thermotoga maritima: the anchor protein OmpA1 (previously termed Ompα) and the porin OmpB (previously termed Ompβ). The gene encoding OmpA (ompA1) was assigned in the genome sequence to TM0477, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. Here we identify the ompB gene as TM0476, determined by LC/MS/MS analysis of the native OmpB protein purified from T. maritima cells. The purified OmpB had β-sheet secondary structure as determined by circular dichroism. Analysis of the sequence of ompB product shows it has porin characteristics including a carboxy terminus anchoring motif and a porin-specific amino acid composition. Orthologs of ompB were found in the genomes of some, but not all, Thermotogales. Those without orthologs have putative analogs. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one to three 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.

  5. 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

  6. 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

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

    2016-03-01

    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

  7. Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

    PubMed

    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

    2016-02-23

    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-cross-linking with [(125)I]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

  8. A Multiple Decrement Life Table Reveals That Host Plant Resistance and Parasitism Are Major Causes of Mortality for the Wheat Stem Sawfly.

    PubMed

    Buteler, Micaela; Peterson, Robert K D; Hofland, Megan L; Weaver, David K

    2015-12-01

    This study investigated the dynamics of parasitism, host plant resistance, pathogens, and predation on the demography of wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), developing in susceptible (hollow stem) and resistant (solid stem) wheat hosts. This study is also the first to investigate the prevalence and impact of cannibalism on wheat stem sawfly mortality. Wheat stem sawflies were sampled in two commercial wheat fields over 4 yr from the egg stage through adult emergence, and multiple decrement life tables were constructed and analyzed. Cannibalism, host plant resistance, or unknown factors were the most prevalent factors causing egg mortality. Summer mortality of prediapause larvae ranged from 28 to 84%, mainly due to parasitism by Bracon cephi (Gahan) and Bracon lissogaster Muesebeck, cannibalism, and host plant resistance. Winter mortality ranged from 6 to 54% of the overwintering larvae, mainly due to unknown factors or pathogens. Cannibalism is a major cause of irreplaceable mortality because it is absolute, with only a single survivor in every multiple infested stem. Subsequent to obligate cannibalism, mortality of feeding larvae due to host plant resistance was lower in hollow stem wheat than in solid stem wheat. Mortality from host plant resistance was largely irreplaceable. Irreplaceable mortality due to parasitoids was greater in hollow stem wheat than in solid stem wheat. Host plant resistance due to stem solidness and parasitism in hollow stems cause substantial mortality in populations of actively feeding larvae responsible for all crop losses. Therefore, enhancing these mortality factors is vital to effective integrated pest management of wheat stem sawfly. PMID:26314030

  9. 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. PMID

  10. 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.

  11. Comparative genome-wide analysis reveals that Burkholderia contaminans MS14 possesses multiple antimicrobial biosynthesis genes but not major genetic loci required for pathogenesis.

    PubMed

    Deng, Peng; Wang, Xiaoqiang; Baird, Sonya M; Showmaker, Kurt C; Smith, Leif; Peterson, Daniel G; Lu, Shien

    2016-06-01

    Burkholderia contaminans MS14 shows significant antimicrobial activities against plant and animal pathogenic fungi and bacteria. The antifungal agent occidiofungin produced by MS14 has great potential for development of biopesticides and pharmaceutical drugs. However, the use of Burkholderia species as biocontrol agent in agriculture is restricted due to the difficulties in distinguishing between plant growth-promoting bacteria and the pathogenic bacteria. The complete MS14 genome was sequenced and analyzed to find what beneficial and virulence-related genes it harbors. The phylogenetic relatedness of B. contaminans MS14 and other 17 Burkholderia species was also analyzed. To research MS14's potential virulence, the gene regions related to the antibiotic production, antibiotic resistance, and virulence were compared between MS14 and other Burkholderia genomes. The genome of B. contaminans MS14 was sequenced and annotated. The genomic analyses reveal the presence of multiple gene sets for antimicrobial biosynthesis, which contribute to its antimicrobial activities. BLAST results indicate that the MS14 genome harbors a large number of unique regions. MS14 is closely related to another plant growth-promoting Burkholderia strain B. lata 383 according to the average nucleotide identity data. Moreover, according to the phylogenetic analysis, plant growth-promoting species isolated from soils and mammalian pathogenic species are clustered together, respectively. MS14 has multiple antimicrobial activity-related genes identified from the genome, but it lacks key virulence-related gene loci found in the pathogenic strains. Additionally, plant growth-promoting Burkholderia species have one or more antimicrobial biosynthesis genes in their genomes as compared with nonplant growth-promoting soil-isolated Burkholderia species. On the other hand, pathogenic species harbor multiple virulence-associated gene loci that are not present in nonpathogenic Burkholderia species. The MS14

  12. 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

  13. Hederagenin, a major component of Clematis mandshurica Ruprecht root, attenuates inflammatory responses in RAW 264.7 cells and in mice.

    PubMed

    Lee, Chul Won; Park, Sang Mi; Zhao, Rongjie; Lee, Chu; Chun, Wonjoo; Son, Yonghae; Kim, Sung Hun; Jung, Ji Yun; Jegal, Kyung Hwan; Cho, Il Je; Ku, Sae Kwang; Kim, Young Woo; Ju, Seong A; Kim, Sang Chan; An, Won G

    2015-12-01

    Clematis mandshurica Ruprecht root has been used in Asia as a traditional anti-inflammatory, analgesic, and antitumor agent. Its main active component is hederagenin, a naturally occurring triterpene, and in this study, we examined the anti-inflammatory effects of hederagenin in lipopolysaccharide-stimulated RAW 264.7 cells using an enzyme-linked immunosorbent assay, Western blot, and RT-PCR. In addition, its effects on acute inflammation in vivo were observed using a carrageenan-induced mouse hind paw edema assay. Furthermore, the changes on the histopathology and histomorphometry of hind paw skins were examined using carrageenan-treated mice. Treatment with hederagenin (10, 30 and 100μM) resulted in inhibited levels of protein expression of lipopolysaccharide-stimulated iNOS, COX-2, and NF-κB as well as production of NO, PGE2, TNF-α, IL-1β, and IL-6 induced by lipopolysaccharide. Consistent with these results, hederagenin also dose-dependently reduced the lipopolysaccharide-induced mRNA levels of iNOS and COX-2, and of the above-mentioned cytokines. Interestingly, results of the carrageenan-induced mouse hind paw edema assay showed an anti-edema effect of hederagenin. Furthermore, hederagenin (30mg/kg) inhibited the carrageenan-induced increases in skin thicknesses, infiltrated inflammatory cells, and mast cell degranulation. These results suggest that hederagenin may possess anti-inflammatory activities. PMID:26481049

  14. 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

  15. 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. PMID:22090315

  16. Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant.

    PubMed

    Gärdby, Eva; Wrammert, Jens; Schön, Karin; Ekman, Lena; Leanderson, Tomas; Lycke, Nils

    2003-01-01

    In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved. PMID:12496383

  17. Pathology of aging female SENCAR mice used as controls in skin two-stage carcinogenesis studies.

    PubMed Central

    Ward, J M; Quander, R; Devor, D; Wenk, M L; Spangler, E F

    1986-01-01

    The pathology of 60 aged female SENCAR mice used as acetone controls in skin painting studies was studied. Fifty percent of the mice survived past 96 weeks of age. The major contributing causes of death identified in 42 mice were glomerulonephritis (8 mice), histiocytic sarcoma (7 mice), and other tumors (8 mice). Glomerulonephritis was found in the majority of mice and was associated with thymic hyperplasia, focal vasculitis, and lymphoid hyperplasia. Necropsy of 58 mice surviving past 50 weeks of age revealed that 41 had an average of 1.36 tumors per mouse. The most common tumors included histiocytic sarcoma (13 mice), pulmonary adenoma or adenocarcinoma (11 mice), mammary tumors (11 mice), follicular center cell lymphoma (4 mice), and hepatocellular adenoma (4 mice). The 13 histiocytic sarcomas appeared to arise in the uterus and metastasized to liver (9 mice), lung (4 mice), kidney (3 mice), and other tissues. Lung tumors were of the solid and papillary types, and tumor cells frequently contained surfactant apoprotein (SAP) but did not contain Clara cell antigens, suggesting their origin from alveolar Type II cells. A variety of nonneoplastic lesions, similar to those observed in other mouse strains, were seen in other tissues of these mice. Amyloid-like material was seen only in nasal turbinates and thyroid gland. In a group of 28 mice exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 88 weeks, as a control for other treatment groups, 7 (25%) had papillomas and 5 (17.8%) had squamous cell carcinomas of the skin at necropsy, although many other induced papillomas regressed during the study. Images FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 13. FIGURE 14. PMID:3780636

  18. Introduction of the six major genomic deletions of modified vaccinia virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice.

    PubMed

    Meisinger-Henschel, Christine; Späth, Michaela; Lukassen, Susanne; Wolferstätter, Michael; Kachelriess, Heike; Baur, Karen; Dirmeier, Ulrike; Wagner, Markus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2010-10-01

    Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions. PMID:20668072

  19. Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse

    PubMed Central

    Ippolito, Gregory C.; Hoi, Kam Hon; Reddy, Sai T.; Carroll, Sean M.; Ge, Xin; Rogosch, Tobias; Zemlin, Michael; Shultz, Leonard D.; Ellington, Andrew D.; VanDenBerg, Carla L.; Georgiou, George

    2012-01-01

    Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors

  20. Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety.

    PubMed

    Briassoulis, George; Keil, Margaret F; Naved, Bilal; Liu, Sophie; Starost, Matthew F; Nesterova, Maria; Gokarn, Nirmal; Batistatos, Anna; Wu, T John; Stratakis, Constantine A

    2016-07-01

    Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA's main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α's function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a(+/-)) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca(+/-)) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a(+/-)/Prkaca(+/-)) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) compared to WT mice. In the EPM, Prkar1a(+/-) spent significantly less time in the open arms, while Prkaca(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain. PMID:26992826

  1. Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Tushima, Anmi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Kuhara, Tomiko; Ohse, Morimasa; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Kobayashi, Keiko

    2011-12-01

    The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency. PMID:21908222

  2. ENU-induced Mutation in the DNA-binding Domain of KLF3 Reveals Important Roles for KLF3 in Cardiovascular Development and Function in Mice

    PubMed Central

    Kelsey, Lois; Flenniken, Ann M.; Qu, Dawei; Funnell, Alister P. W.; Pearson, Richard; Zhou, Yu-Qing; Voronina, Irina; Berberovic, Zorana; Wood, Geoffrey; Newbigging, Susan; Weiss, Edward S.; Wong, Michael; Quach, Ivan; Yeh, S. Y. Sandy; Deshwar, Ashish R.; Scott, Ian C.; McKerlie, Colin; Henkelman, Mark; Backx, Peter; Simpson, Jeremy; Osborne, Lucy; Rossant, Janet; Crossley, Merlin; Bruneau, Benoit; Adamson, S. Lee

    2013-01-01

    KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3H275R). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3H275R mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3H275R mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans. PMID:23874215

  3. Major depression

    MedlinePlus

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

  4. The type III transporters (PiT-1 and PiT-2) are the major sodium-dependent phosphate transporters in the mice and human brains.

    PubMed

    Inden, Masatoshi; Iriyama, Masaki; Zennami, Miho; Sekine, Shin-Ichiro; Hara, Akira; Yamada, Mitsunori; Hozumi, Isao

    2016-04-15

    PiT-1/SLC20A1 and PiT-2/SLC20A2 are members of the mammalian type-III inorganic phosphate (Pi) transporters encoded by the SLC20 genes. The broad distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues is compatible with housekeeping maintenace of intracellular Pi homeostasis by transporting Pi from intrastitial fluid for normal cellular functions. Recently, mutations of SLC20A2 have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease. However, the localization of PiT-1 and PiT-2 in the normal brain has not been clarified yet. The aim of this study was to reveal the distribution of PiT-1 and PiT-2 in the mouse and human brains. As results, gene expressing analysis showed that SLC20A1 and SLC20A2 mRNAs were widely expressed throughout the mouse and human brains, although other Pi transporters encoded by SLC17 and SLC34 mRNAs were hardly detected. The region of cerebellum contained a higher level of SLC20A1 and SLC20A2 mRNAs than the other brain regions. Additionally, the cerebellum in the mouse brain contained higher levels of PiT-1 and PiT-2 than those in the other regions in the brain, respectively. The immonohistochemical studies showed that PiT-1 was recognized in neuron, astrocytes and vascular endothelial cells. Similarly to PiT-1 immunopositivity, PiT-2 was clearly recognized in these cells. These results suggest that SLC20 family plays a pivotal role in the maintenance of cellular Pi homeostasis particullary in the brain. The viewpoint is compatible with the finding that calcification in IBGC is recognized only in the brain. This provides us with a novel viewpoint to understand the basic pathophysiology of IBGC through type-III Pi transporters. PMID:26923164

  5. 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. PMID:27470339

  6. 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.

  7. Skeletal Muscle MicroRNA and Messenger RNA Profiling in Cofilin-2 Deficient Mice Reveals Cell Cycle Dysregulation Hindering Muscle Regeneration

    PubMed Central

    Morton, Sarah U.; Joshi, Mugdha; Savic, Talia; Beggs, Alan H.; Agrawal, Pankaj B.

    2015-01-01

    Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2) have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies. PMID:25874796

  8. Generation of Mice Deficient in both KLF3/BKLF and KLF8 Reveals a Genetic Interaction and a Role for These Factors in Embryonic Globin Gene Silencing

    PubMed Central

    Funnell, Alister P. W.; Mak, Ka Sin; Twine, Natalie A.; Pelka, Gregory J.; Norton, Laura J.; Radziewic, Tania; Power, Melinda; Wilkins, Marc R.; Bell-Anderson, Kim S.; Fraser, Stuart T.; Perkins, Andrew C.; Tam, Patrick P.; Pearson, Richard C. M.

    2013-01-01

    Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development. PMID:23716600

  9. Role of the postnatal radial glial scaffold for the development of the dentate gyrus as revealed by Reelin signaling mutant mice

    PubMed Central

    Brunne, Bianka; Franco, Santos; Bouché, Elisabeth; Herz, Joachim; Howell, Brian W.; Pahle, Jasmine; Müller, Ulrich; May, Petra; Frotscher, Michael; Bock, Hans H.

    2014-01-01

    During dentate gyrus development the early embryonic radial glial scaffold is replaced by a secondary glial scaffold around birth. In contrast to neocortical and early dentate gyrus radial glial cells these postnatal glial cells are severely altered with regard to position and morphology in reeler mice lacking the secreted protein Reelin. In this study we focus on the functional impact of these defects. Most radial glial cells throughout the nervous system serve as scaffolds for migrating neurons and precursor cells for both neurogenesis and gliogenesis. Precursor cell function has been demonstrated for secondary radial glial cells but the exact function of these late glial cells in granule cell migration and positioning is not clear. No data exist concerning the interplay between granule neurons and late radial glial cells during dentate gyrus development. Here we show that despite the severe morphological defects in the reeler dentate gyrus the precursor function of secondary radial glial cells is not impaired during development in reeler mice. In addition, selective ablation of Disabled-1, an intracellular adaptor protein essential for Reelin signaling, in neurons but not in glial cells allowed us to distinguish effects of Reelin signaling on radial glial cells from possible secondary effects based on defective granule cells positioning. PMID:23828756

  10. 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!

  11. Dysfunction in Ribosomal Gene Expression in the Hypothalamus and Hippocampus following Chronic Social Defeat Stress in Male Mice as Revealed by RNA-Seq

    PubMed Central

    Smagin, Dmitry A.; Kovalenko, Irina L.; Galyamina, Anna G.; Bragin, Anatoly O.; Orlov, Yuriy L.; Kudryavtseva, Natalia N.

    2016-01-01

    Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs, and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with “ribosomopathies.” PMID:26839715

  12. Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle

    PubMed Central

    Coopersmith, Craig M.; Chandrasekaran, Chitra; McNevin, M. Shane; Gordon, Jeffrey I.

    1997-01-01

    Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ∼30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 × TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108→ Lys107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 × TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a

  13. Dysfunction in Ribosomal Gene Expression in the Hypothalamus and Hippocampus following Chronic Social Defeat Stress in Male Mice as Revealed by RNA-Seq.

    PubMed

    Smagin, Dmitry A; Kovalenko, Irina L; Galyamina, Anna G; Bragin, Anatoly O; Orlov, Yuriy L; Kudryavtseva, Natalia N

    2016-01-01

    Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs, and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with "ribosomopathies." PMID:26839715

  14. Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

    PubMed

    Rindi, G; Ratineau, C; Ronco, A; Candusso, M E; Tsai, M; Leiter, A B

    1999-09-01

    The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells. PMID:10457023

  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. PMID:24688298

  18. 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

  19. Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice.

    PubMed

    Assem, Mahfoud; Schuetz, Erin G; Leggas, Markos; Sun, Daxi; Yasuda, Kazuto; Reid, Glen; Zelcer, Noam; Adachi, Masashi; Strom, Stephen; Evans, Ronald M; Moore, David D; Borst, Piet; Schuetz, John D

    2004-05-21

    The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver. PMID:15004017

  20. 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. PMID:25592253

  1. Ultrahigh sensitive optical microangiography reveals depth-resolved microcirculation and its longitudinal response to prolonged ischemic event within skeletal muscles in mice

    NASA Astrophysics Data System (ADS)

    Jia, Yali; Qin, Jia; Zhi, Zhongwei; Wang, Ruikang K.

    2011-08-01

    The primary pathophysiology of peripheral arterial disease is associated with impaired perfusion to the muscle tissue in the lower extremities. The lack of effective pharmacologic treatments that stimulate vessel collateralization emphasizes the need for an imaging method that can be used to dynamically visualize depth-resolved microcirculation within muscle tissues. Optical microangiography (OMAG) is a recently developed label-free imaging method capable of producing three-dimensional images of dynamic blood perfusion within microcirculatory tissue beds at an imaging depth of up to ~2 mm, with an unprecedented imaging sensitivity of blood flow at ~4 μm/s. In this paper, we demonstrate the utility of OMAG in imaging the detailed blood flow distributions, at a capillary-level resolution, within skeletal muscles of mice. By use of the mouse model of hind-limb ischemia, we show that OMAG can assess the time-dependent changes in muscle perfusion and perfusion restoration along tissue depth. These findings indicate that OMAG can represent a sensitive, consistent technique to effectively study pharmacologic therapies aimed at promoting the growth and development of collateral vessels.

  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. 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.

  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. High fat diet induced alterations of atrial electrical activities in mice

    PubMed Central

    Zhang, Fan; Hartnett, Sigurd; Sample, Alex; Schnack, Sabrina; Li, Yifan

    2016-01-01

    Obesity is a well-known risk factor for various cardiovascular diseases. Recent clinical data showed that overweight and obese patients have higher incidence of atrial fibrillation (AF) compared with individuals with normal body weights, but the underlying mechanisms remain to be elucidated. In this study, we investigated the effects of a high fat diet on atrial activities in mice. ICR male mice were fed a regular diet (RD) or a high fat diet (HFD) for 8 weeks. Mice fed HFD showed significantly greater body weight gains and visceral fat accumulation compared with RD mice. Under anesthetic condition, baseline arterial blood pressure and heart rate were not significantly different between RD and HFD groups. Although no spontaneous or atrial stimulation-induced atrial fibrillation was observed, this study revealed several alterations in the activities and protein levels in the atria in HFD mice. Surface electrocardiogram (ECG) revealed significantly shortened PR interval in HFD mice. In the atrial stimulation experiments, the sinoatrial (SA) node recovery time was significantly prolonged whereas the atrial effective refractory period was significantly reduced in HFD mice as compared with RD mice. Western blot showed protein levels of two major potassium channels, Kv1.5 and Kv4.2/3, were significantly increased in atria of HFD mice. These data indicate that HFD induces atrial electrophysiological remodeling in mice, which may be a potential mechanism underlying the increased risk for atrial arrhythmias in obesity and metabolic disorders. PMID:27073731

  6. Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging

    PubMed Central

    Masuda, Tadashi; Croom, Deborah

    2013-01-01

    the fraction of beaded dendrites that were incapable to recover during repolarization, thus facilitating terminal injury. In contrast, similarly induced four rounds of spreading depolarization in another set of control healthy mice caused no accumulating dendritic injury as dendrites fully recovered from beading during repolarization. Taken together, our data suggest that in the mild traumatic brain injury the acute dendritic injury in the peri-contusional cortex is gated by the decline in the local blood flow, most probably as a result of developing oedema. Furthermore, spreading depolarization is a specific mechanism that could accelerate injury to synaptic circuitry in the metabolically compromised peri-contusional cortex, worsening secondary damage following traumatic brain injury. PMID:23466395

  7. Dissecting lipid metabolism in meibomian glands of humans and mice: An integrative study reveals a network of metabolic reactions not duplicated in other tissues.

    PubMed

    Butovich, Igor A; McMahon, Anne; Wojtowicz, Jadwiga C; Lin, Feng; Mancini, Ronald; Itani, Kamel

    2016-06-01

    Lipids comprise the bulk of the meibomian gland secretion (meibum) which is produced by meibocytes. Complex arrays of lipogenic reactions in meibomian glands, which we collectively call meibogenesis, have not been explored on a molecular level yet. Our goals were to elucidate the possible biosynthetic pathways that underlie the generation of meibum, reveal similarities in, and differences between, lipid metabolism in meibomian glands and other organs and tissues, and integrate meibomian gland studies into the field of general metabolomics. Specifically, we have conducted detailed analyses of human and mouse specimens using genomic, immunohistochemical, and lipidomic approaches. Among equally highly expressed genes found in meibomian glands of both species were those related to fatty acid elongation, branching, desaturation, esterification, reduction of fatty acids to alcohols, and cholesterol biosynthesis. Importantly, corresponding lipid products were detected in meibum of both species using lipidomic approaches. For the first time, a cohesive, unifying biosynthetic scheme that connects genomic, lipidomic, and immunohistochemical observations is outlined and discussed. PMID:27032494

  8. 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)

  9. Endothelial dysfunction of cerebral and major arteries during chronic obstructive disease.

    PubMed

    Geltser, B I; Brodskaya, T A; Kotelnikov, V N; Agafonova, I G; Lukyanov, P A

    2007-12-01

    Vasomotor activity of the major and cerebral arteries was studied in mice with chronic obstructive pulmonary disease. Regional differences were revealed in the endothelium-dependent response of arteries. The development of chronic obstructive pulmonary disease was associated with a paradoxical response of the dilatational component of vasoregulation against the background of increased constrictive influences of the vascular endothelium in the major and cerebral vessels. PMID:18856197

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

    PubMed

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

    2016-01-01

    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

  11. Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

    PubMed

    Trachtulec, Z; Hamvas, R M; Forejt, J; Lehrach, H R; Vincek, V; Klein, J

    1997-08-15

    The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates. PMID:9286694

  12. Of mice and (Viking?) men: phylogeography of British and Irish house mice.

    PubMed

    Searle, Jeremy B; Jones, Catherine S; Gündüz, Islam; Scascitelli, Moira; Jones, Eleanor P; Herman, Jeremy S; Rambau, R Victor; Noble, Leslie R; Berry, R J; Giménez, Mabel D; Jóhannesdóttir, Fríoa

    2009-01-22

    The west European subspecies of house mouse (Mus musculus domesticus) has gained much of its current widespread distribution through commensalism with humans. This means that the phylogeography of M. m. domesticus should reflect patterns of human movements. We studied restriction fragment length polymorphism (RFLP) and DNA sequence variations in mouse mitochondrial (mt) DNA throughout the British Isles (328 mice from 105 localities, including previously published data). There is a major mtDNA lineage revealed by both RFLP and sequence analyses, which is restricted to the northern and western peripheries of the British Isles, and also occurs in Norway. This distribution of the 'Orkney' lineage fits well with the sphere of influence of the Norwegian Vikings and was probably generated through inadvertent transport by them. To form viable populations, house mice would have required large human settlements such as the Norwegian Vikings founded. The other parts of the British Isles (essentially most of mainland Britain) are characterized by house mice with different mtDNA sequences, some of which are also found in Germany, and which probably reflect both Iron Age movements of people and mice and earlier development of large human settlements. MtDNA studies on house mice have the potential to reveal novel aspects of human history. PMID:18826939

  13. 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. PMID:25134869

  14. 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…

  15. 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

  16. Crybb2 deficiency impairs fertility in female mice

    SciTech Connect

    Gao, Qian; Sun, Li-Li; Xiang, Fen-Fen; Gao, Li; Jia, Yin; Zhang, Jian-Rong; Tao, Hai-Bo; Zhang, Jun-Jie; Li, Wen-Jie

    2014-10-10

    Highlights: • Crybb2 deletion impaired female fertility. • Crybb2 deletion dramatically affected the production of reproduction-related hormones and hormone response. • Crybb2 deletion impaired follicular development and inhibited the proliferation of granulosa cells. • Crybb2 deletion promoted follicular atresia and apoptosis in granulosa cells. - Abstract: Beta-B2-crystallin (CRYBB2), encoded by Crybb2 gene, is a major protein in the mammalian eye lens that plays an important role in maintaining the transparency of the ocular lens. However, CRYBB2 also plays important roles in many extra-lenticular tissues and organs such as the retina, brain and testis. Our previous studies demonstrated that male Crybb2 deficient (Crybb2{sup −/−}) mice have reduced fertility compared with wild-type (WT) mice, while female Crybb2{sup −/−} mice exhibited reduced ovary weights and shorter estrous cycle percentages. Here we specifically investigated the role of CRYBB2 in the female reproductive system. Our studies revealed that ovaries from female Crybb2{sup −/−} mice exhibited significantly reduced numbers of primordial, secondary and pre-ovulatory follicles when compared with WT mice, while the rate of atretic follicles was also increased. Additionally, fewer eggs were collected from the oviduct of Crybb2{sup −/−} female mice after superovulation. Estrogen levels were higher in the metestrus and diestrus cycles of female Crybb2{sup −/−} mice, while progesterone levels were lower in diestrus cycles. Furthermore, the expression of survival and cell cycle genes, Bcl-2, Cdk4 and Ccnd2, were significantly decreased in granulosa cells isolated from female Crybb2{sup −/−} mice, consistent with the predominant expression of CRYBB2 in ovarian granulosa cells. Our results reveal a critical role for CRYBB2 in female fertility and specific effects on the proliferation and survival status of ovarian granulosa cells.

  17. Syrtis Major

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site]

    Released 18 May 2004 This image of Syrtis Major was acquired August 20, 2002, during northern spring.

    The THEMIS VIS camera is capable of capturing color images of the martian surface using its five different color filters. In this mode of operation, the spatial resolution and coverage of the image must be reduced to accommodate the additional data volume produced from the use of multiple filters. To make a color image, three of the five filter images (each in grayscale) are selected. Each is contrast enhanced and then converted to a red, green, or blue intensity image. These three images are then combined to produce a full color, single image. Because the THEMIS color filters don't span the full range of colors seen by the human eye, a color THEMIS image does not represent true color. Also, because each single-filter image is contrast enhanced before inclusion in the three-color image, the apparent color variation of the scene is exaggerated. Nevertheless, the color variation that does appear is representative of some change in color, however subtle, in the actual scene. Note that the long edges of THEMIS color images typically contain color artifacts that do not represent surface variation.

    Image information: VIS instrument. Latitude 12.8, Longitude 79.5 East (280.5 West). 38 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The

  18. Carboxypeptidase N-deficient mice present with polymorphic disease phenotypes on induction of experimental autoimmune encephalomyelitis.

    PubMed

    Hu, Xianzhen; Wetsel, Rick A; Ramos, Theresa N; Mueller-Ortiz, Stacey L; Schoeb, Trenton R; Barnum, Scott R

    2014-02-01

    Carboxypeptidase N (CPN) is a member of the carboxypeptidase family of enzymes that cleave carboxy-terminal lysine and arginine residues from a large number of biologically active peptides and proteins. These enzymes are best known for their roles in modulating the activity of kinins, complement anaphylatoxins and coagulation proteins. Although CPN makes important contributions to acute inflammatory events, little is known about its role in autoimmune disease. In this study we used CPN(-/-) mice in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Unexpectedly, we observed several EAE disease phenotypes in CPN(-/-) mice compared to wild type mice. The majority of CPN(-/-) mice died within five to seven days after disease induction, before displaying clinical signs of disease. The remaining mice presented with either mild EAE or did not develop EAE. In addition, CPN(-/-) mice injected with complete or incomplete Freund's adjuvant died within the same time frame and in similar numbers as those induced for EAE. Overall, the course of EAE in CPN(-/-) mice was significantly delayed and attenuated compared to wild type mice. Spinal cord histopathology in CPN(-/-) mice revealed meningeal, but not parenchymal leukocyte infiltration, and minimal demyelination. Our results indicate that CPN plays an important role in EAE development and progression and suggests that multiple CPN ligands contribute to the disease phenotypes we observed. PMID:24028840

  19. Genetic Labeling of Steroidogenic Factor-1 (SF-1) Neurons in Mice Reveals Ventromedial Nucleus of the Hypothalamus (VMH) Circuitry Beginning at Neurogenesis and Development of a Separate Non-SF-1 Neuronal Cluster in the Ventrolateral VMH

    PubMed Central

    Cheung, Clement C.; Kurrasch, Deborah M.; Liang, Jenna K.; Ingraham, Holly A.

    2015-01-01

    The ventromedial nucleus of the hypothalamus (VMH) influences a wide variety of physiological responses. Here, using two distinct but complementary genetic tracing approaches in mice, we describe the development of VMH efferent projections, as marked by steroidogenic factor-1 (SF-1; NR5A1). SF-1 neurons were visualized by Tau-green fluorescent protein (GFP) expressed from the endogenous Sf-1 locus (Sf-1TauGFP) or by crossing the transgenic Sf1:Cre driver to a GFP reporter strain (Z/EGSf1:Cre). Strikingly, VMH projections were visible early, at embryonic (E) 10.5, when few postmitotic SF1 neurons have been born, suggesting that formation of VMH circuitry begins at the onset of neurogenesis. At E14.5, comparison of these two reporter lines revealed that SF1-positive neurons in the ventrolateral VMH (VMHvl) persist in Z/EGSf1:Cre embryos but are virtually absent in Sf-1TauGFP. Therefore, although the entire VMH including the VMHvl shares a common lineage, the VMHvl further differentiates into a neuronal cluster devoid of SF-1. At birth, extensive VMH projections to broad regions of the brain were observed in both mouse reporter lines, matching well with those previously discovered by injection of axonal anterograde tracers in adult rats. In summary, our genetic tracing studies show that VMH efferent projections are highly conserved in rodents and are established far earlier than previously appreciated. Moreover, our results imply that neurons in the VMHvl adopt a distinct fate early in development, which might underlie the unique physiological functions associated with this VMH subregion. PMID:22987798

  20. MICE Construction Status

    SciTech Connect

    Zisman, Michael S.

    2008-02-21

    MICE, the Muon Ionization Cooling Experiment, is an approved experiment at the Rutherford Appleton Laboratory. In this paper, we briefly review the aims of the experiment, give an overview of the system, and indicate the design and/or construction status of the various major subsystems that comprise the experiment. First beam is expected in January 2008.

  1. Non-motor behavioural impairments in parkin-deficient mice.

    PubMed

    Zhu, Xin-Ran; Maskri, Lyutha; Herold, Christina; Bader, Verian; Stichel, Christine C; Güntürkün, Onur; Lübbert, Hermann

    2007-10-01

    Mutations in the parkin gene are the major cause of early-onset familial Parkinson's disease (PD). We previously reported the generation and analysis of a knockout mouse carrying a deletion of exon 3 in the parkin gene. F1 hybrid pa+/- mice were backcrossed to wild-type C57Bl/6 for three more generations to establish a pa-/-(F4) mouse line. The appearance of tyrosine hydroxylase-positive neurons was normal in young and aged pa-/- (F4) animals. Loss of parkin function in mice did not enhance vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. However, the pa-/- (F4) mice displayed impaired exploration and habituation to a new environment and exhibited thigmotaxis behaviour in the open field and Morris water maze. Abnormal anxiety-related behaviour of pa-/- (F4) mice was also observed in the light/dark exploration test paradigm. Dopamine metabolism was enhanced in the striatum of pa-/- (F4) mice, as revealed by increased homovanillic acid (HVA) content and a reduced ratio of dihydroxyphenylacetic acid (DOPAC)/HVA. The alterations found in the dopaminergic system could be responsible for the behavioural impairments of pa-/- (F4) mice. Consistent with a recent observation of cognitive dysfunction in parkin-linked patients with PD, our findings provide evidence of a physiological role of parkin in non-motor behaviour, possibly representing a disease stage that precedes dopaminergic neuron loss. PMID:17883413

  2. A Novel Intronic Single Nucleotide Polymorphism in the Myosin heavy polypeptide 4 Gene Is Responsible for the Mini-Muscle Phenotype Characterized by Major Reduction in Hind-Limb Muscle Mass in Mice

    PubMed Central

    Kelly, Scott A.; Bell, Timothy A.; Selitsky, Sara R.; Buus, Ryan J.; Hua, Kunjie; Weinstock, George M.; Garland, Theodore; Pardo-Manuel de Villena, Fernando; Pomp, Daniel

    2013-01-01

    Replicated artificial selection for high levels of voluntary wheel running in an outbred strain of mice favored an autosomal recessive allele whose primary phenotypic effect is a 50% reduction in hind-limb muscle mass. Within the High Runner (HR) lines of mice, the numerous pleiotropic effects (e.g., larger hearts, reduced total body mass and fat mass, longer hind-limb bones) of this hypothesized adaptive allele include functional characteristics that facilitate high levels of voluntary wheel running (e.g., doubling of mass-specific muscle aerobic capacity, increased fatigue resistance of isolated muscles, longer hind-limb bones). Previously, we created a backcross population suitable for mapping the responsible locus. We phenotypically characterized the population and mapped the Minimsc locus to a 2.6-Mb interval on MMU11, a region containing ∼100 known or predicted genes. Here, we present a novel strategy to identify the genetic variant causing the mini-muscle phenotype. Using high-density genotyping and whole-genome sequencing of key backcross individuals and HR mice with and without the mini-muscle mutation, from both recent and historical generations of the HR lines, we show that a SNP representing a C-to-T transition located in a 709-bp intron between exons 11 and 12 of the Myosin heavy polypeptide 4 (Myh4) skeletal muscle gene (position 67,244,850 on MMU11; assembly, December 2011, GRCm38/mm10; ENSMUSG00000057003) is responsible for the mini-muscle phenotype, Myh4Minimsc. Using next-generation sequencing, our approach can be extended to identify causative mutations arising in mouse inbred lines and thus offers a great avenue to overcome one of the most challenging steps in quantitative genetics. PMID:24056412

  3. Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

    PubMed Central

    Seyed, Negar; Taheri, Tahereh; Vauchy, Charline; Dosset, Magalie; Godet, Yann; Eslamifar, Ali; Sharifi, Iraj; Adotevi, Olivier; Borg, Christophe; Rohrlich, Pierre Simon; Rafati, Sima

    2014-01-01

    Background There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II. Methods and Findings HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(51Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential. Conclusions Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine

  4. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus).

    PubMed

    Kim, Kwang-Hwan; Hwang, Ji-Hyun; Han, Dong-Yeup; Park, Minkyu; Kim, Seungill; Choi, Doil; Kim, Yongjae; Lee, Gung Pyo; Kim, Sun-Tae; Park, Young-Hoon

    2015-01-01

    An intraspecific genetic map for watermelon was constructed using an F2 population derived from 'Arka Manik' × 'TS34' and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits. PMID:26700647

  5. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus)

    PubMed Central

    Kim, Kwang-Hwan; Hwang, Ji-Hyun; Han, Dong-Yeup; Park, Minkyu; Kim, Seungill; Choi, Doil; Kim, Yongjae; Lee, Gung Pyo; Kim, Sun-Tae; Park, Young-Hoon

    2015-01-01

    An intraspecific genetic map for watermelon was constructed using an F2 population derived from ‘Arka Manik’ × ‘TS34’ and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits. PMID:26700647

  6. Comparative Analyses Between Lolium/Festuca Introgression Lines and Rice Reveal the Major Fraction of Functionally Annotated Gene Models Is Located in Recombination-Poor/Very Recombination-Poor Regions of the Genome

    PubMed Central

    King, Julie; Armstead, Ian P.; Donnison, S. Iain; Roberts, Luned A.; Harper, John A.; Skøt, Kirsten; Elborough, Kieran; King, Ian P.

    2007-01-01

    Publication of the rice genome sequence has allowed an in-depth analysis of genome organization in a model monocot plant species. This has provided a powerful tool for genome analysis in large-genome unsequenced agriculturally important monocot species such as wheat, barley, rye, Lolium, etc. Previous data have indicated that the majority of genes in large-genome monocots are located toward the ends of chromosomes in gene-rich regions that undergo high frequencies of recombination. Here we demonstrate that a substantial component of the coding sequences in monocots is localized proximally in regions of very low and even negligible recombination frequencies. The implications of our findings are that during domestication of monocot plant species selection has concentrated on genes located in the terminal regions of chromosomes within areas of high recombination frequency. Thus a large proportion of the genetic variation available for selection of superior plant genotypes has not been exploited. In addition our findings raise the possibility of the evolutionary development of large supergene complexes that confer a selective advantage to the individual. PMID:17603095

  7. Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses.

    PubMed Central

    Okahashi, N; Yamamoto, M; Vancott, J L; Chatfield, S N; Roberts, M; Bluethmann, H; Hiroi, T; Kiyono, H; McGhee, J R

    1996-01-01

    Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses. PMID:8613355

  8. Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

    PubMed

    Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

    2013-08-01

    The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases. PMID:23855516

  9. Whole DNA methylome profiling in mice exposed to secondhand smoke

    PubMed Central

    Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

    2012-01-01

    Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease. PMID:23051858

  10. Acute and chronic nephrotoxicity of platinum nanoparticles in mice

    NASA Astrophysics Data System (ADS)

    Yamagishi, Yoshiaki; Watari, Akihiro; Hayata, Yuya; Li, Xiangru; Kondoh, Masuo; Yoshioka, Yasuo; Tsutsumi, Yasuo; Yagi, Kiyohito

    2013-09-01

    Platinum nanoparticles are being utilized in various industrial applications, including in catalysis, cosmetics, and dietary supplements. Although reducing the size of the nanoparticles improves the physicochemical properties and provides useful performance characteristics, the safety of the material remains a major concern. The aim of the present study was to evaluate the biological effects of platinum particles less than 1 nm in size (snPt1). In mice administered with a single intravenous dose of snPt1, histological analysis revealed necrosis of tubular epithelial cells and urinary casts in the kidney, without obvious toxic effects in the lung, spleen, and heart. These mice exhibited dose-dependent elevation of blood urea nitrogen, an indicator of kidney damage. Direct application of snPt1 to in vitro cultures of renal cells induced significant cytotoxicity. In mice administered for 4 weeks with twice-weekly intraperitoneal snPt1, histological analysis of the kidney revealed urinary casts, tubular atrophy, and inflammatory cell accumulation. Notably, these toxic effects were not observed in mice injected with 8-nm platinum particles, either by single- or multiple-dose administration. Our findings suggest that exposure to platinum particles of less than 1 nm in size may induce nephrotoxicity and disrupt some kidney functions. However, this toxicity may be reduced by increasing the nanoparticle size.

  11. Postnatal Hematopoiesis and Gut Microbiota in NOD Mice Deviate from C57BL/6 Mice

    PubMed Central

    Damlund, Dina Silke Malling; Metzdorff, Stine Broeng; Hasselby, Jane Preuss; Wiese, Maria; Lundsager, Mia; Buschard, Karsten Stig; Hansen, Axel Kornerup; Frøkiær, Hanne

    2016-01-01

    Neonatal studies in different mouse strains reveal that early life colonization affects the development of adaptive immunity in mice. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes, but neonatal studies of NOD mice are lacking. We hypothesized that NOD mice deviate from another much used mouse strain, C57BL/6, with respect to postnatal microbiota and/or hematopoiesis and compared this in newborn mice of dams housed under the same conditions. A distinct bacteria profile rich in staphylococci was found at postnatal days (PND) 1–4 in NOD mice. Furthermore, a distinct splenic cell profile high in a granulocytic phenotype was evident in the neonatal NOD mice whereas neonatal C57BL/6 mice showed a profile rich in monocytes. Neonatal expression of Reg3g and Muc2 in the gut was deviating in NOD mice and coincided with fewer bacteria attaching to the Mucosal surface in NOD compared to C57BL/6 mice. PMID:26783537

  12. Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background

    PubMed Central

    Bleich, Andre; Sundberg, John P; Smoczek, Anna; von Wasielewski, Reinhard; de Buhr, Maike F; Janus, Lydia M; Julga, Gwen; Ukena, Sya N; Hedrich, Hans-J; Gunzer, Florian

    2008-01-01

    Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium’s probiotic nature should be the focus of further studies. PMID:18005134

  13. Cardiac hypertrophy associated with myeloproliferative neoplasms in JAK2V617F transgenic mice

    PubMed Central

    2014-01-01

    Background Myeloproliferative neoplasms (MPNs) are blood malignancies manifested in increased production of red blood cells, white blood cells, and/or platelets. A major molecular lesion associated with the diseases is JAK2V617F, an activation mutation form of tyrosine kinase JAK2. Cardiovascular events represent the leading cause of morbidity and mortality associated MPNs, but the underlying mechanism is not well understood. Methods Previously, we generated JAK2V617F transgenic mice which displayed MPN-like phenotypes. In the present study, we further characterized these mice by analyzing the time course of MPN phenotype development and associated cardiac abnormalities. We performed detailed histochemical staining of cardiac sections. Results JAK2V617F transgenic mice developed cardiomegaly as a subsequent event of increased blood cell production during the course of MPN phenotype development. The cardiomegaly is manifested in increased ventricular wall thickness and enlarged cardiomyocytes. Trichrome and reticulin staining revealed extensive collagen fibrosis in the heart of JAK2V617F transgenic mice. Thrombosis in the coronary artery and inflammatory cell infiltration into cardiac muscle were also observed in JAK2V617F transgenic mice, and the latter event was accompanied by fibrosis. Conclusion JAK2V617F-induced blood disorders have a major impact on heart function and lead to cardiac hypertrophy. JAK2V617F transgenic mice represent an excellent model system to study both hematological malignancies and cardiovascular diseases. PMID:24646493

  14. Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP(5+), in plasma and major organs of B6C3F1 mice.

    PubMed

    Spasojević, Ivan; Chen, Yumin; Noel, Teresa J; Fan, Ping; Zhang, Lichun; Rebouças, Julio S; St Clair, Daret K; Batinić-Haberle, Ines

    2008-10-01

    Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined. Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed, with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters

  15. Impact of clock gene Bmal1 deficiency on nutritionally induced obesity in mice.

    PubMed

    Hemmeryckx, Bianca; Himmelreich, Uwe; Hoylaerts, Marc F; Lijnen, Henri R

    2011-03-01

    To evaluate the hypothesis that the clock gene Bmal1 (brain and muscle arnt like protein-1) plays a role in the development of obesity, 5-week-old male Bmal1-deficient (Bmal1(-/-)) mice and wild-type littermates (Bmal1(+/+)) were kept on a high-fat diet (HFD) for 15 weeks. Despite an initial accelerated weight gain of Bmal1(-/-) mice, body weight and subcutaneous (SC) and gonadal (GON) adipose tissue mass were comparable to Bmal1(+/+) mice at the end of the diet period. Noninvasive magnetic resonance imaging scanning revealed a modest increase in fat content in Bmal1(-/-) mice after 10 weeks of HFD, whereas at the start and the end of the HFD feeding no differences were observed between both genotypes. After 15 weeks of HFD, adipocyte and blood vessel size and density were similar for Bmal1(+/+) and Bmal1(-/-) mice. However, the weight of major organs was significantly reduced in Bmal1(-/-) mice, confirming the premature ageing phenotype. Thus, we hypothesize that an initial accelerated increase in body weight and fat mass of Bmal1(-/-) mice on HFD may have been offset by the effect of premature ageing on organ weight, resulting in comparable weights after 15 weeks of HFD. PMID:21030946

  16. Phenotyping by magnetic resonance imaging nondestructively measures glomerular number and volume distribution in mice with and without nephron reduction

    PubMed Central

    Baldelomar, Edwin J.; Charlton, Jennifer R.; Beeman, Scott C.; Hann, Bradley D.; Cullen-McEwen, Luise; Pearl, Valeria M.; Bertram, John F.; Wu, Teresa; Zhang, Min; Bennett, Kevin M.

    2015-01-01

    Reduced nephron mass is strongly linked to susceptibility to chronic renal and cardiovascular diseases. There are currently no tools to identify nephropenia in clinical or preclinical diagnostics. Such new methods could uncover novel mechanisms and therapies for chronic kidney disease (CKD) and reveal how variation among traits can affect renal function and morphology. Here we used cationized ferritin (CF) enhanced-MRI (CFE-MRI) to investigate the relationship between glomerular number (Nglom) and volume (Vglom) in kidneys of healthy wild type mice and mice with oligosyndactylism (Os/+), a model of congenital nephron reduction. Mice were injected with cationic ferritin and perfused and the resected kidneys imaged with 7T MRI to detect CF-labeled glomeruli. CFE-MRI was used to measure the intrarenal distribution of individual glomerular volumes and revealed two major populations of glomeruli distinguished by size. Spatial mapping revealed that the largest glomeruli were located in the juxtamedullary region in both wild type and Os/+ mice and the smallest population located in the cortex. Os/+ mice had about a 50% reduction and 35% increase of Nglom and Vglom, respectively, in both glomerular populations compared to wild type, consistent with glomerular hypertrophy in the Os/+ mice. Thus, we provide a foundation for whole-kidney, MRI-based phenotyping of mouse renal glomerular morphology and provide new potential for quantitative human renal diagnostics. PMID:26535998

  17. Phenotyping by magnetic resonance imaging nondestructively measures glomerular number and volume distribution in mice with and without nephron reduction.

    PubMed

    Baldelomar, Edwin J; Charlton, Jennifer R; Beeman, Scott C; Hann, Bradley D; Cullen-McEwen, Luise; Pearl, Valeria M; Bertram, John F; Wu, Teresa; Zhang, Min; Bennett, Kevin M

    2016-02-01

    Reduced nephron mass is strongly linked to susceptibility to chronic renal and cardiovascular diseases. There are currently no tools to identify nephropenia in clinical or preclinical diagnostics. Such new methods could uncover novel mechanisms and therapies for chronic kidney disease (CKD) and reveal how variation among traits can affect renal function and morphology. Here we used cationized ferritin (CF)–enhanced MRI (CFE-MRI) to investigate the relationship between glomerular number (Nglom) and volume (Vglom) in kidneys of healthy wild-type mice and mice with oligosyndactylism (Os/+), a model of congenital nephron reduction. Mice were injected with CF and perfused, and the resected kidneys were imaged with 7T MRI to detect CF-labeled glomeruli. CFE-MRI was used to measure the intrarenal distribution of individual glomerular volumes and revealed two major populations of glomeruli distinguished by size. Spatial mapping revealed that the largest glomeruli were located in the juxtamedullary region in both wild-type and Os/+ mice and the smallest population located in the cortex. Os/+ mice had about a 50% reduction and 35% increase of Nglom and Vglom, respectively, in both glomerular populations compared with wild type, consistent with glomerular hypertrophy in the Os/+ mice. Thus, we provide a foundation for whole-kidney, MRI-based phenotyping of mouse renal glomerular morphology and provide new potential for quantitative human renal diagnostics. PMID:26535998

  18. Genetic Analysis of T Cell Lymphomas in Carbon Ion-Irradiated Mice Reveals Frequent Interstitial Chromosome Deletions: Implications for Second Cancer Induction in Normal Tissues during Carbon Ion Radiotherapy.

    PubMed

    Blyth, Benjamin J; Kakinuma, Shizuko; Sunaoshi, Masaaki; Amasaki, Yoshiko; Hirano-Sakairi, Shinobu; Ogawa, Kanae; Shirakami, Ayana; Shang, Yi; Tsuruoka, Chizuru; Nishimura, Mayumi; Shimada, Yoshiya

    2015-01-01

    Monitoring mice exposed to carbon ion radiotherapy provides an indirect method to evaluate the potential for second cancer induction in normal tissues outside the radiotherapy target volume, since such estimates are not yet possible from historical patient data. Here, male and female B6C3F1 mice were given single or fractionated whole-body exposure(s) to a monoenergetic carbon ion radiotherapy beam at the Heavy Ion Medical Accelerator in Chiba, Japan, matching the radiation quality delivered to the normal tissue ahead of the tumour volume (average linear energy transfer = 13 keV x μm(-1)) during patient radiotherapy protocols. The mice were monitored for the remainder of their lifespan, and a large number of T cell lymphomas that arose in these mice were analysed alongside those arising following an equivalent dose of 137Cs gamma ray-irradiation. Using genome-wide DNA copy number analysis to identify genomic loci involved in radiation-induced lymphomagenesis and subsequent detailed analysis of Notch1, Ikzf1, Pten, Trp53 and Bcl11b genes, we compared the genetic profile of the carbon ion- and gamma ray-induced tumours. The canonical set of genes previously associated with radiation-induced T cell lymphoma was identified in both radiation groups. While the pattern of disruption of the various pathways was somewhat different between the radiation types, most notably Pten mutation frequency and loss of heterozygosity flanking Bcl11b, the most striking finding was the observation of large interstitial deletions at various sites across the genome in carbon ion-induced tumours, which were only seen infrequently in the gamma ray-induced tumours analysed. If such large interstitial chromosomal deletions are a characteristic lesion of carbon ion irradiation, even when using the low linear energy transfer radiation to which normal tissues are exposed in radiotherapy patients, understanding the dose-response and tissue specificity of such DNA damage could prove key to

  19. Genetic Analysis of T Cell Lymphomas in Carbon Ion-Irradiated Mice Reveals Frequent Interstitial Chromosome Deletions: Implications for Second Cancer Induction in Normal Tissues during Carbon Ion Radiotherapy

    PubMed Central

    Blyth, Benjamin J.; Kakinuma, Shizuko; Sunaoshi, Masaaki; Amasaki, Yoshiko; Hirano-Sakairi, Shinobu; Ogawa, Kanae; Shirakami, Ayana; Shang, Yi; Tsuruoka, Chizuru; Nishimura, Mayumi; Shimada, Yoshiya

    2015-01-01

    Monitoring mice exposed to carbon ion radiotherapy provides an indirect method to evaluate the potential for second cancer induction in normal tissues outside the radiotherapy target volume, since such estimates are not yet possible from historical patient data. Here, male and female B6C3F1 mice were given single or fractionated whole-body exposure(s) to a monoenergetic carbon ion radiotherapy beam at the Heavy Ion Medical Accelerator in Chiba, Japan, matching the radiation quality delivered to the normal tissue ahead of the tumour volume (average linear energy transfer = 13 keV.μm-1) during patient radiotherapy protocols. The mice were monitored for the remainder of their lifespan, and a large number of T cell lymphomas that arose in these mice were analysed alongside those arising following an equivalent dose of 137Cs gamma ray-irradiation. Using genome-wide DNA copy number analysis to identify genomic loci involved in radiation-induced lymphomagenesis and subsequent detailed analysis of Notch1, Ikzf1, Pten, Trp53 and Bcl11b genes, we compared the genetic profile of the carbon ion- and gamma ray-induced tumours. The canonical set of genes previously associated with radiation-induced T cell lymphoma was identified in both radiation groups. While the pattern of disruption of the various pathways was somewhat different between the radiation types, most notably Pten mutation frequency and loss of heterozygosity flanking Bcl11b, the most striking finding was the observation of large interstitial deletions at various sites across the genome in carbon ion-induced tumours, which were only seen infrequently in the gamma ray-induced tumours analysed. If such large interstitial chromosomal deletions are a characteristic lesion of carbon ion irradiation, even when using the low linear energy transfer radiation to which normal tissues are exposed in radiotherapy patients, understanding the dose-response and tissue specificity of such DNA damage could prove key to assessing

  20. Soluble maltase-glucoamylase is alternatively spliced and secreted by Paneth and goblet cells in enterocyte maltase-glucoamylase Null mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is the major energy source in the mouse diet. Knockout of intestinal membrane-bound maltase-glucoamylase (Mgam) in mice revealed presence of soluble starch digesting activity in Null jejunum and ileum that is secreted by Paneth and goblet cells. Hypotheses: 1. WT have two Mgam mRNAs, Mgamme...

  1. Alterations in Oral [1-14C] 18:1n-9 Distribution in Lean Wild-Type and Genetically Obese (ob/ob) Mice

    PubMed Central

    Wang, Xinxia; Feng, Jie; Yu, Caihua; Shen, Qingwu W.; Wang, Yizhen

    2015-01-01

    Obesity may result from altered fatty acid (FA) disposal. Altered FA distribution in obese individuals is poorly understood. Lean wild-type C57BL/6J and obese C57BL/6Job/ob mice received an oral dose of [1-14C]18:1n-9 (oleic acid), and the radioactivity in tissues was evaluated at various time points. The 14C concentration decreased rapidly in gastrointestinal tract but gradually increased and peaked at 96 h in adipose tissue, muscle and skin in lean mice. The 14C concentration was constant in adipose tissue and muscle of obese mice from 4h to 168h. 14C-label content in adipose tissue was significantly affected by genotype, whereas muscle 14C-label content was affected by genotype, time and the interaction between genotype and time. There was higher total 14C retention (47.7%) in obese mice than in lean mice (9.0%) at 168 h (P<0.05). The 14C concentrations in the soleus and gastrocnemius muscle were higher in obese mice than in lean mice (P<0.05). Perirenal adipose tissue contained the highest 14C content in lean mice, whereas subcutaneous adipose tissue (SAT) had the highest 14C content and accounted for the largest proportion of total radioactivity among fat depots in obese mice. More lipid radioactivity was recovered as TAG in SAT from obese mice than from lean mice (P<0.05). Gene expression suggested acyl CoA binding protein and fatty acid binding protein are important for FA distribution in adipose tissue and muscle. The FA distribution in major tissues was altered in ob/ob mice, perhaps contributing to obesity. Understanding the disparity in FA disposal between lean and obese mice may reveal novel targets for the treatment and prevention of obesity. PMID:25826747

  2. Type I interferon signaling protects mice from lethal henipavirus infection.

    PubMed

    Dhondt, Kévin P; Mathieu, Cyrille; Chalons, Marie; Reynaud, Joséphine M; Vallve, Audrey; Raoul, Hervé; Horvat, Branka

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are closely related, recently emerged paramyxoviruses that form Henipavirus genus and are capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. However, in contrast to many other species and despite expression of functional virus entry receptors, mice are resistant to henipavirus infection. We report here the susceptibility of mice deleted for the type I interferon receptor (IFNAR-KO) to both HeV and NiV. Intraperitoneally infected mice developed fatal encephalitis, with pathology and immunohistochemical features similar to what was found in humans. Viral RNA was found in the majority of analyzed organs, and sublethally infected animals developed virus-specific neutralizing antibodies. Altogether, these results reveal IFNAR-KO mice as a new small animal model to study HeV and NiV pathogenesis, prophylaxis, and treatment and suggest the critical role of type I interferon signaling in the control of henipavirus infection. PMID:23089589

  3. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  4. Marginal Maternal Zinc Deficiency in Lactating Mice Reduces Secretory Capacity and Alters Milk Composition12

    PubMed Central

    Dempsey, Colleen; McCormick, Nicholas H.; Croxford, Thomas P.; Seo, Young Ah; Grider, Arthur; Kelleher, Shannon L.

    2012-01-01

    Dietary analysis predicts that marginal Zn deficiency is common in women of reproductive age. The lack of reliable biomarkers limits the capacity to assess Zn status and consequently understand effects of maternal Zn deficiency. We determined effects of marginal maternal Zn deficiency on mammary gland function, milk secretion, and milk composition in mice. Mice (n = 12/diet) were fed marginal (ZD; 15 mg Zn/kg diet) or adequate (ZA; 30 mg Zn/kg diet) Zn diets for 30 d prior to conception through mid-lactation. Mice fed the ZD had a higher plasma Zn concentration (~20%; P < 0.05) but lower milk Zn concentration (~15%; P < 0.05) compared with mice fed the ZA. ZnT2 abundance was higher (P < 0.05) in mice fed the ZD compared with mice fed the ZA; no effect on ZnT4 abundance was detected. The Zn concentration of mammary gland mitochondria tended to be ~40% greater in mice fed ZD (P = 0.07); this was associated with apoptosis and lower milk secretion (~80%; P < 0.01). Total milk protein was ~25% higher (P < 0.05), although the abundance of the major milk proteins (caseins and whey acidic protein) was lower (P < 0.05) in mice fed the ZD. Proteomic analysis of milk proteins revealed an increase (P < 0.05) in four proteins in mice fed the ZD. These findings illustrate that marginal maternal Zn deficiency compromises mammary gland function and milk secretion and alters milk composition. This suggests that lactating women who consume inadequate Zn may not produce and/or secrete an adequate amount of high quality milk to provide optimal nutrition to their developing infant. PMID:22357740

  5. Rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages.

    PubMed

    Nagarkar, Deepti R; Bowman, Emily R; Schneider, Dina; Wang, Qiong; Shim, Jee; Zhao, Ying; Linn, Marisa J; McHenry, Christina L; Gosangi, Babina; Bentley, J Kelley; Tsai, Wan C; Sajjan, Umadevi S; Lukacs, Nicholas W; Hershenson, Marc B

    2010-08-15

    Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-gamma. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations. PMID:20644177

  6. It's Major! College Major Selection & Success

    ERIC Educational Resources Information Center

    Byers, Jenny; Mattern, Krista D.; Shaw, Emily J.; Springall, Robert

    2011-01-01

    Presented at the College Board National Forum, October 26, 2011. Choosing a college major is challenging enough, without stopping to consider the impact it has on a student's college experience and career choice. To provide support during this major decision, participants in this session will develop strategies to facilitate students in making an…

  7. Grass pollen hypersensitivity in mice

    PubMed Central

    McCaskill, A. C.; Hosking, C. S.; Hill, D. J.

    1982-01-01

    Mice were sensitized by intranasal administration of ryegrass pollen. Subsequent nasal challenge with pollen extract led to a `shock' response peaking in severity 4 hr after challenge. Histological examination of lungs revealed the development of a pneumonitis which was most severe 3 days after challenge. ImagesFigure 2 PMID:7106842

  8. Morphological, physiological and behavioural evaluation of a 'Mice in Space' housing system.

    PubMed

    Blottner, Dieter; Serradj, Najet; Salanova, Michele; Touma, Chadi; Palme, Rupert; Silva, Mitchell; Aerts, Jean Marie; Berckmans, Daniel; Vico, Laurence; Liu, Yi; Giuliani, Alessandra; Rustichelli, Franco; Cancedda, Ranieri; Jamon, Marc

    2009-05-01

    Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called "Mice in Space" (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories. PMID:19130060

  9. Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

    PubMed

    Thelemann, Christoph; Haller, Sergio; Blyszczuk, Przemyslaw; Kania, Gabriela; Rosa, Muriel; Eriksson, Urs; Rotman, Samuel; Reith, Walter; Acha-Orbea, Hans

    2016-03-01

    Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis. PMID:26621778

  10. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  11. Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

    PubMed

    Burvenich, Ingrid J G; Farrugia, William; Lee, Fook T; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J; Brechbiel, Martin W; King, Dylan; Spirkoska, Violeta; Allan, Laura C; Ramsland, Paul A; Scott, Andrew M

    2016-01-01

    IgG has a long half-life through engage