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Sample records for mouse express n-acyl

  1. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner.

    PubMed

    Erikson, Elina; Wratil, Paul R; Frank, Martin; Ambiel, Ina; Pahnke, Katharina; Pino, Maria; Azadi, Parastoo; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Meier, Chris; Schnaar, Ronald L; Crocker, Paul R; Reutter, Werner; Keppler, Oliver T

    2015-11-06

    Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.

  2. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner*

    PubMed Central

    Erikson, Elina; Wratil, Paul R.; Frank, Martin; Ambiel, Ina; Pahnke, Katharina; Pino, Maria; Azadi, Parastoo; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Meier, Chris; Schnaar, Ronald L.; Crocker, Paul R.; Reutter, Werner; Keppler, Oliver T.

    2015-01-01

    Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction. PMID:26370074

  3. Exogenous N-acyl-homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants.

    PubMed

    Cheng, Feifei; Ma, Anzhou; Zhuang, Guoqiang; Fray, Rupert G

    2016-10-18

    In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N-acyl-homoserine lactone (AHL)-mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL-producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL-producing plants compared with wild-type plants. The present data indicate that plant-produced AHLs enhance disease resistance against this pathogen. Subsequent RNA-sequencing analysis showed that the exogenous addition of AHLs up-regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL-producing and wild-type plants were determined by quantitative real-time polymerase chain reaction. These data showed that plant-produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant-produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.

  4. Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii

    PubMed Central

    Modarresi, Farzan; Azizi, Omid; Shakibaie, Mohammad Reza; Motamedifar, Mohammad; Mansouri, Shahla

    2016-01-01

    Background and Objectives: In present study we aimed to clone the luxI gene encoding N-acyl-homoserine synthase detected in clinical isolates of Acinetobacter baumannii and study its expression in Escherichia coli transformants. Materials and Methods: Four A. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. The presence of luxI gene was detected using PCR technique. Purified PCR product DNA was initially cloned into pTG19 and transformed to E. coli DH5α. The gene was then recovered from agarose gel and ligated by T4 DNA ligase into pET28a expression vector using NdeI and XhoI enzymes. pET28a + luxI was transformed into E. coli BL21 (DE3). The luxI putative gene was further detected in the transformants by colony PCR. Expression of the luxI gene in the recombinant E. coli BL21 cells was studied by quantitative real time PCR (qRT-PCR) and the presence of N-acylhomoserine lactone (AHL) was checked by colorimetric assay and Fourier Transform Infra-Red (FT-IR) spectroscopy. Results: We successfully cloned AHL gene from A. baumannii strain 23 to pET28a expression vector. There was four fold increases in expression of luxI in the transformants (P ≤ 0.05). It was found that, strain 23 and the transformants showed highest amount of AHL activity (OD = 1.524). The FT-IR analysis indicated stretching C=O bond of the lactone ring and primary amides (N=H) at 1764.69 cm−1 and 1659.23 cm−1 respectively. Conclusion: From above results we concluded that, luxI in A. baumannii is indeed responsible for AHL production and not regulation and pET28a vector allows efficient AHL expression in E. coli BL21 transformants. PMID:27307980

  5. Quorum Sensing N-acyl Homoserine Lactones-SdiA Suppresses Escherichia coli-Pseudomonas aeruginosa Conjugation through Inhibiting traI Expression

    PubMed Central

    Lu, Yang; Zeng, Jianming; Wu, Binning; E, Shunmei; Wang, Lina; Cai, Renxin; Zhang, Ni; Li, Youqiang; Huang, Xianzhang; Huang, Bin; Chen, Cha

    2017-01-01

    Conjugation is a key mechanism for horizontal gene transfer and plays an important role in bacterial evolution, especially with respect to antibiotic resistance. However, little is known about the role of donor and recipient cells in regulation of conjugation. Here, using an Escherichia coli (SM10λπ)-Pseudomonas aeruginosa (PAO1) conjugation model, we demonstrated that deficiency of lasI/rhlI, genes associated with generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or deletion of the AHLs receptor SdiA in the donor SM10λπ both facilitated conjugation. When using another AHLs-non-producing E. coli strain EC600 as recipient cells, deficiency of sdiA in donor SM10λπ hardly affect the conjugation. More importantly, in the presence of exogenous AHLs, the conjugation efficiency between SM10λπ and EC600 was dramatically decreased, while deficiency of sdiA in SM10λπ attenuated AHLs-inhibited conjugation. These data suggest the conjugation suppression function of AHLs-SdiA chemical signaling. Further bioinformatics analysis, β-galactosidase reporter system and electrophoretic mobility shift assays characterized the binding site of SdiA on the promoter region of traI gene. Furthermore, deletion of lasI/rhlI or sdiA promoted traI mRNA expression in SM10λπ and PAO1 co-culture system, which was abrogated by AHLs. Collectively, our results provide new insight into an important contribution of quorum sensing system AHLs-SdiA to the networks that regulate conjugation. PMID:28164039

  6. Metabolic Glycoengineering with N-Acyl Side Chain Modified Mannosamines.

    PubMed

    Wratil, Paul R; Horstkorte, Rüdiger; Reutter, Werner

    2016-08-08

    In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N-acyl-modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman-Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N-acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE).

  7. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines

    PubMed Central

    Ogura, Yuji; Parsons, William H.; Kamat, Siddhesh S.; Cravatt, Benjamin F.

    2016-01-01

    More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells. PMID:27399000

  8. Endogenous N-acyl taurines regulate skin wound healing

    PubMed Central

    Sasso, Oscar; Pontis, Silvia; Armirotti, Andrea; Cardinali, Giorgia; Kovacs, Daniela; Migliore, Marco; Summa, Maria; Moreno-Sanz, Guillermo; Picardo, Mauro; Piomelli, Daniele

    2016-01-01

    The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs—N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]—as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy. PMID:27412859

  9. Copper(II)/amine synergistically catalyzed enantioselective alkylation of cyclic N-acyl hemiaminals with aldehydes.

    PubMed

    Sun, Shutao; Mao, Ying; Lou, Hongxiang; Liu, Lei

    2015-07-07

    The first catalytic asymmetric alkylation of N-acyl quinoliniums with aldehydes has been described. A copper/amine synergistic catalytic system has been developed, allowing the addition of functionalized aldehydes to a wide range of electronically varied N-acyl quinoliniums in good yields with excellent enantiocontrol. The synergistic catalytic system was also effective for N-acyl dihydroisoquinoliniums and β-caboliniums, demonstrating the general applicability of the protocol in the enantioselective alkylation of diverse cyclic N-acyl hemiaminals.

  10. Thiourea-Catalyzed Aminolysis of N-acyl Homoserine Lactones

    DTIC Science & Technology

    2013-01-01

    of N-acyl homoserine lactones (AHLs), molecules integral to bacterial quorum sensing . The catalysts afford rate enhancement of up to 10 times the...SUBJECT TERMS quorum sensing Michael A. Bertucci, Stephen J. Lee, Michel R. Gagné University of North Carolina - Chapel Hill Office of Sponsored... quorum sensing . The catalysts afford rate enhancement of up to 10 times the control in CD3CN. Mild catalysis in other polar aprotic solvents is

  11. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

    Imker, Heidi J.; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T.

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  12. AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

    PubMed

    See-Too, Wah Seng; Ee, Robson; Lim, Yan-Lue; Convey, Peter; Pearce, David A; Yin, Wai-Fong; Chan, Kok-Gan

    2017-02-22

    Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15(T), isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.

  13. AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

    PubMed Central

    See-Too, Wah Seng; Ee, Robson; Lim, Yan-Lue; Convey, Peter; Pearce, David A.; Yin, Wai-Fong; Chan, Kok-Gan

    2017-01-01

    Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions. PMID:28225085

  14. Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

    PubMed

    Rivera, Patricia; Arrabal, Sergio; Vargas, Antonio; Blanco, Eduardo; Serrano, Antonia; Pavón, Francisco J; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

  15. Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

    PubMed Central

    Rivera, Patricia; Arrabal, Sergio; Vargas, Antonio; Blanco, Eduardo; Serrano, Antonia; Pavón, Francisco J.; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca2+ fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca2+-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα+/calbindin+ cells were closely surrounded by NAPE-PLD+ fiber varicosities. No pyramidal PPARα+/calbindin+ cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD+/calretinin+ cells were specifically detected in CA3. NAPE-PLD+ puncta surrounded the calretinin+ cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions. PMID:24672435

  16. Inhibiting N-acyl-homoserine lactone synthesis and quenching Pseudomonas quinolone quorum sensing to attenuate virulence

    PubMed Central

    Chan, Kok-Gan; Liu, Yi-Chia; Chang, Chien-Yi

    2015-01-01

    Bacteria sense their own population size, tune the expression of responding genes, and behave accordingly to environmental stimuli by secreting signaling molecules. This phenomenon is termed as quorum sensing (QS). By exogenously manipulating the signal transduction bacterial population behaviors could be controlled, which may be done through quorum quenching (QQ). QS related regulatory networks have been proven their involvement in regulating many virulence determinants in pathogenic bacteria in the course of infections. Interfering with QS signaling system could be a novel strategy against bacterial infections and therefore requires more understanding of their fundamental mechanisms. Here we review the development of studies specifically on the inhibition of production of N-acyl-homoserine lactone (AHL), a common proteobacterial QS signal. The opportunistic pathogen, Pseudomonas aeruginosa, equips the alkylquinolone (AQ)-mediated QS which also plays crucial roles in its pathogenicity. The studies in QQ targeting on AQ are also discussed. PMID:26539190

  17. New N-acyl taurine from the sea urchin Glyptocidaris crenularis.

    PubMed

    Zhou, Xuefeng; Xu, Tunhai; Wen, Kewei; Yang, Xian-Wen; Xu, Shi-Hai; Liu, Yonghong

    2010-01-01

    A new N-acyl taurine (1), together with a new natural product, l-(beta-D-ribofuranosyl)-1,2,4-triazole (4), and two known compounds (2 and 3), were isolated from the sea urchin, Glyptocidaris crenularis. The new N-acyl taurine was elucidated as 2-(5R,15S-dihydroxyeicosanoylamino) ethanesulfonic acid on the basis of spectroscopic (NMR, MS) analyses and the modified Mosher ester method. Compound 2 showed significant toxicity against brine shrimp larvae.

  18. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (surfactants) at levels not to exceed 10% in pesticide formulations containing glyphosate: Name CAS Reg. No. N... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  19. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (surfactants) at levels not to exceed 10% in pesticide formulations containing glyphosate: Name CAS Reg. No. N... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  20. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  1. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  2. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  3. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    PubMed Central

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  4. Modified N-acyl-homoserine lactones as chemical probes for the elucidation of plant-microbe interactions.

    PubMed

    Thomanek, Heike; Schenk, Sebastian T; Stein, Elke; Kogel, Karl-Heinz; Schikora, Adam; Maison, Wolfgang

    2013-09-25

    Gram-negative bacteria often use N-acyl-homoserine lactones (AHLs) as signal molecules to monitor their local population densities and to regulate gene-expression in a process called "Quorum Sensing" (QS). This cell-to-cell communication allows bacteria to adapt to environmental changes and to behave as multicellular communities. QS plays a key role in both bacterial virulence towards the host and symbiotic interactions with other organisms. Plants also perceive AHLs and respond to them with changes in gene expression or modifications in development. Herein, we report the synthesis of new AHL-derivatives for the investigation and identification of AHL-interacting proteins. We show that our new compounds are still recognised by different bacteria and that a novel biotin-tagged-AHL derivative interacts with a bacterial AHL receptor.

  5. Thermoregulation of N-acyl homoserine lactone-based quorum sensing in the soft rot bacterium Pectobacterium atrosepticum.

    PubMed

    Latour, Xavier; Diallo, Stéphanie; Chevalier, Sylvie; Morin, Danièle; Smadja, Bruno; Burini, Jean-François; Haras, Dominique; Orange, Nicole

    2007-06-01

    The psychrotolerant bacterium Pectobacterium atrosepticum produces four N-acyl homoserine lactones under a wide range of temperatures. Their thermoregulation differs from that of the exoenzyme production, described as being under quorum-sensing control. A mechanism involved in this thermoregulation consists of controlling N-acyl homoserine lactones synthase production at a transcriptional level.

  6. N-acyl homoserinelactone-mediated gene regulation in gram-negative bacteria.

    PubMed

    Eberl, L

    1999-12-01

    The view of bacteria as unicellular organisms has strong roots in the tradition of culturing bacteria in liquid media. However, in nature microbial activity is mainly associated with surfaces where bacteria form highly structured and cooperative consortia which are commonly referred to as biofilms. The ability of bacteria to organize structurally and to distribute metabolic activities between the different members of the consortium demands a high degree of coordinated cell-cell interaction. Recent work has established that many bacteria employ sophisticated intercellular communication systems that rely on small signal molecules to control the expression of multiple target genes. In Gram-negative bacteria, the most intensively investigated signal molecules are N-acyl-L-homoserine lactones (AHLs), which are utilized by the bacteria to monitor their own population densities in a process known as 'quorum sensing'. These density-dependent regulatory systems rely on two proteins, an AHL synthase, usually a member of the LuxI family of proteins, and an AHL receptor protein belonging to the LuxR family of transcriptional regulators. At low population densities cells produce a basal level of AHL via the activity of an AHL synthase. As the cell density increases, AHL accumulates in the growth medium. On reaching a critical threshold concentration, the AHL molecule binds to its cognate receptor which in turn leads to the induction/repression of AHL-regulated genes. To date, AHL-dependent quorum sensing circuits have been identified in a wide range of gram-negative bacteria where they regulate various functions including bioluminescence, plasmid conjugal transfer, biofilm formation, motility, antibiotic biosynthesis, and the production of virulence factors in plant and animal pathogens. Moreover, AHL signal molecules appear to play important roles in the ecology of complex consortia as they allow bacterial populations to interact with each other as well as with their

  7. N-Acyl-phosphoramidates as potential novel form of gemcitabine prodrugs.

    PubMed

    Baraniak, Janina; Pietkiewicz, Aleksandra; Kaczmarek, Renata; Radzikowska, Ewa; Kulik, Katarzyna; Krolewska, Karolina; Cieslak, Marcin; Krakowiak, Agnieszka; Nawrot, Barbara

    2014-04-01

    Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency. Thus, proper prodrugs carrying the 5'-pdFdC moiety may help to overcome this problem. A series of new derivatives of gemcitabine possessing N-acyl(thio)phosphoramidate moieties were prepared and their cytotoxic properties were determined. N-Acyl-phosphoramidate derivatives of gemcitabine have similar cytotoxicity as gemcitabine itself, and have been found accessible to the cellular enzymes. The nicotinic carboxamide derivative of gemcitabine 5'-O-phosphorothioate occurred to be the best inhibitor of bacterial DNA polymerase I and human DNA polymerase α.

  8. Synthesis, Surface Active Properties and Cytotoxicity of Sodium N-Acyl Prolines.

    PubMed

    Sreenu, Madhumanchi; Narayana Prasad, Rachapudi Badari; Sujitha, Pombala; Kumar, Chityal Ganesh

    2015-01-01

    Sodium N-acyl prolines (NaNAPro) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 58-75% yields to study the synergetic effect of mixture of hydrophobic fatty acyl functionalities like saturation, unsaturation and cyclopropene fatty acids with different chain lengths and aliphatic hetero cyclic proline head group on their surface and cytotoxicity activities. The products were characterized by chromatographic and spectral techniques. The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results revealed that all the products exhibited superior surface active properties like CMC, calcium tolerance and emulsion stability as compared to the standard surfactant, sodium lauryl sulphate (SLS). In addition, palm, Sterculia foetida and high oleic sunflower fatty N-acyl prolines exhibited promising cytotoxicity against different tumor cell lines.

  9. Expression of mouse metallothionein genes in tobacco

    SciTech Connect

    Maiti, I.B.; Yeargan, R.; Wagner, G.J.; Hunt, A.G. )

    1990-05-01

    We have expressed a mouse metallothionein (NT) gene in tobacco under control of the cauliflower mosaic virus (CaMV) 35S promoter and a pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoter. Seedlings in which MT gene expression is driven by the 35S promoter are resistant to toxic levels of cadmium. Mature plants carrying the 35S-MT gene accumulate less Cd in their leaves when exposed to low levels of Cd in laboratory growth conditions. Plants with the rbcS-MT construction express this gene in a light-regulated and tissue-specific manner, as expected. Moreover, the MT levels in leaves in these plants are about 20% of those seen in 35S-MT plants. These plants are currently being tested for Cd resistance. In addition, a small field evaluation of 35S-MT lines for Cd levels is being evaluated. These experiments will address the possibility of using MTs to alter Cd levels in crop species.

  10. Arylpiperazines with N-acylated amino acids as 5-HT1A receptor ligands.

    PubMed

    Zajdel, Paweł; Subra, Gilles; Bojarski, Andrzej J; Duszyńska, Beata; Pawłowski, Maciej; Martinez, Jean

    2006-07-01

    A library consisting of 60 arylpiperazines modified with N-acylated amino acids was prepared on BAL linker SynPhasetrade mark Lanterns and evaluated in vitro for 5-HT(1A) receptor affinity. Biological screening, followed by a simple Fujita-Ban analysis, enabled the description of structure-activity relationships and allowed the selection of some potent, high-affinity ligands for in vivo pharmacological investigations.

  11. Syntheses and Antibacterial Activity of N-Acylated Ciprofloxacin Derivatives Based on the Trimethyl Lock

    PubMed Central

    2015-01-01

    Several N-acyl ciprofloxacin quinone derivatives based on a trimethyl lock structure were synthesized, and their in vitro antibacterial activity against a panel of clinically relevant bacteria was evaluated. A few new analogues displayed enhanced activity against Gram-positive species compared to the parent drug. Additionally, studies of 8-Cip, which was the most potent compound tested, indicate that it may act through a dual-action mechanism. PMID:26101578

  12. Facile synthesis of N-acyl 2-aminobenzothiazoles by NHC-catalyzed direct oxidative amidation of aldehydes.

    PubMed

    Premaletha, Sethulekshmy; Ghosh, Arghya; Joseph, Sumi; Yetra, Santhivardhana Reddy; Biju, Akkattu T

    2017-01-26

    A mild, general, and high yielding synthesis of N-acyl 2-aminobenzothiazoles has been demonstrated by N-heterocyclic carbene (NHC)-organocatalyzed direct amidation of aldehydes with 2-aminobenzothiazoles proceeding via acyl azolium intermediates. The carbene generated from the triazolium salt under oxidative conditions was the key for the success of this reaction. The method was subsequently applied to the synthesis of various biologically important N-acyl 2-aminobenzothiazoles.

  13. Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay for N-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

    PubMed Central

    Last, Daniel; Krüger, Georg H. E.; Dörr, Mark

    2016-01-01

    ABSTRACT Quorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to l-homoserine. In turn, l-homoserine is detected by the previously described calcein assay, which is sensitive to α-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-l-homoserine. IMPORTANCE In this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-l-homoserine lactones. These are the most common

  14. TRPM3 expression in mouse retina.

    PubMed

    Brown, R Lane; Xiong, Wei-Hong; Peters, James H; Tekmen-Clark, Merve; Strycharska-Orczyk, Iwona; Reed, Brian T; Morgans, Catherine W; Duvoisin, Robert M

    2015-01-01

    Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

  15. Ceramide metabolism in mouse tissue.

    PubMed

    Schiffmann, Susanne; Birod, Kerstin; Männich, Julia; Eberle, Max; Wegner, Marthe-Susanna; Wanger, Ruth; Hartmann, Daniela; Ferreiros, Nerea; Geisslinger, Gerd; Grösch, Sabine

    2013-08-01

    Ceramides with different N-acyl chains can act as second messengers in various signaling pathways. They are involved in cell processes such as apoptosis, differentiation and inflammation. Ceramide synthases (CerS) are key enzymes in the biosynthesis of ceramides and dihydroceramides. Six isoenzymes (CerS1-6) catalyze the N-acylation of the sphingoid bases, albeit with strictly acyl-Coenzyme A (CoA) chain length specificity. We analyzed the mRNA expression, the protein expression, the specific activity of the CerS, and acyl-CoA, dihydroceramide and ceramide levels in different tissues by LC-MS/MS. Our data indicate that each tissue express a distinct composition of CerS, whereby the CerS mRNA expression levels do not correlate with the respective protein expression levels in the tissues. Furthermore, we found a highly significant negative correlation between the protein expression level of CerS6 and the C16:0-acyl-CoA amounts as well as between the protein expression of CerS2 and C24:0-acyl-CoA amounts. These data indicate that in mouse tissues low substrate availability is compensated by higher CerS protein expression level and vice versa. Apart from the expression level and the specific activity of the CerS, other enzymes of the sphingolipid pathway also influence the composition of ceramides with distinct chain lengths in each cell. Acyl-CoA availability seems to be less important for ceramide composition and might be compensated for by CerS expression/activity.

  16. Structure, supramolecular organization and phase behavior of N-acyl-β-alanines: Structural homologues of mammalian brain constituents N-acylglycine and N-acyl-GABA.

    PubMed

    Sivaramakrishna, D; Swamy, Musti J

    2016-12-01

    N-Acyl-β-alanines (NABAs) are structural homologues of N-acylglycines (NAGs) and N-acyl-γ-aminobutyric acids (NAGABAs), and achiral isomers of N-acylalanines, which are all present in mammalian brain and other tissues and modulate activity of biological receptors with various functions. In the present study, we synthesized and characterized a homologous series of NABAs bearing saturated acyl chains (n=8-20) and investigated their supramolecular organization and thermotropic phase behavior. In differential scanning calorimetric (DSC) studies, most of the NABAs gave one or two minor transitions before the main chain-melting phase transition in the dry state as well as upon hydration with water, but gave only a single transition when hydrated with buffer (pH7.6). Transition enthalpies (ΔHt) and entropies (ΔSt), obtained from the DSC studies showed linear dependence on the chain length in the dry state and upon hydration with buffer, whereas odd-even alteration was observed when hydrated with water. The crystal structures of N-lauroyl-β-alanine (NLBA) and N-myristoyl-β-alanine (NMBA) were solved in monoclinic system in the P21/c space group. Both NLBA and NMBA were packed in tilted bilayers with head-to-head (and tail-to-tail) arrangement with tilt angles of 33.28° and 34.42°, respectively. Strong hydrogen bonding interactions between COOH groups of the molecules from opposite leaflets as well as NH⋯O hydrogen bonds between the amide groups from adjacent molecules in the same leaflet as well as dispersion interactions between the acyl chains stabilize the bilayer structure. The d-spacings calculated from powder X-ray diffraction studies showed odd-even alteration with odd-chain length compounds exhibiting higher values as compared to the even-chain length ones and the tilt angles calculated from the PXRD data are higher for the even chain NABAs. These observations are relevant to developing structure-activity relationships for these amphiphiles and understand

  17. Orally administered thermostable N-acyl homoserine lactonase from Bacillus sp. strain AI96 attenuates Aeromonas hydrophila infection in zebrafish.

    PubMed

    Cao, Yanan; He, Suxu; Zhou, Zhigang; Zhang, Meichao; Mao, Wei; Zhang, Huitu; Yao, Bin

    2012-03-01

    N-Acylated homoserine lactone (AHL) lactonases are capable of degrading signal molecules involved in bacterial quorum sensing and therefore represent a new approach to control bacterial infection. Here a gene responsible for the AHL lactonase activity of Bacillus sp. strain AI96, 753 bp in length, was cloned and then expressed in Escherichia coli. The deduced amino acid sequence of Bacillus sp. AI96 AiiA (AiiA(AI96)) is most similar to those of other Bacillus sp. AHL lactonases (~80% sequence identity) and was consequently categorized as a member of the metallo-β-lactamase superfamily. AiiA(AI96) maintains ~100% of its activity at 10°C to 40°C at pH 8.0, and it is very stable at 70°C at pH 8.0 for at least 1 h; no other Bacillus AHL lactonase has been found to be stable under these conditions. AiiA(AI96) resists digestion by proteases and carp intestinal juice, and it has broad-spectrum substrate specificity. The supplementation of AiiA(AI96) into fish feed by oral administration significantly attenuated Aeromonas hydrophila infection in zebrafish. This is the first report of the oral administration of an AHL lactonase for the efficient control of A. hydrophila.

  18. A New N-Acyl Homoserine Lactone Synthase in an Uncultured Symbiont of the Red Sea Sponge Theonella swinhoei

    PubMed Central

    Britstein, Maya; Devescovi, Giulia; Handley, Kim M.; Malik, Assaf; Haber, Markus; Saurav, Kumar; Teta, Roberta; Costantino, Valeria; Burgsdorf, Ilia; Gilbert, Jack A.; Sher, Noa; Venturi, Vittorio

    2015-01-01

    Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N-acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei. The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales, here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli, produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium. PMID:26655754

  19. Lipoprotein N-acyl transferase (Lnt1) is dispensable for protein O-mannosylation by Streptomyces coelicolor.

    PubMed

    Córdova-Dávalos, Laura Elena; Espitia, Clara; González-Cerón, Gabriela; Arreguín-Espinosa, Roberto; Soberón-Chávez, Gloria; Servín-González, Luis

    2014-01-01

    A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

  20. Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

    PubMed Central

    Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

    2014-01-01

    A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

  1. Synthesis and Biological Activity of Mono- and Di-N-acylated Aminoglycosides

    PubMed Central

    2015-01-01

    Despite issues with oto/nephrotoxicity and bacterial resistance, aminoglycosides (AGs) remain an effective and widely used class of antibacterial agents. For decades now, efforts toward the development of novel AGs with potential to overcome some of these problems have been major research focuses. 1-N-Acylation, especially γ-amino-β-hydroxybutyrate (AHB) derivatization, has proven to be one of the most successful strategies for improving the overall properties of AGs, including their ability to avoid certain resistance mechanisms. More recently, 6′-N-acylation arose as another possible strategy to improve the properties of these drugs. In this study, we report on the glycinyl, carboxybenzyl, and AHB mono- and diderivatization at the 1-, 6′-, and/or 4‴-amines of the AGs amikacin, kanamycin A, netilmicin, sisomicin, and tobramycin. We also present the antibacterial activities and the reduced reactivity of AG-modifying enzymes (AMEs) toward these new AG derivatives, and identify the AMEs present in the bacterial strains tested. PMID:26617967

  2. Cyclin A1 is expressed in mouse ovary.

    PubMed

    Wei, Hongquan; Li, Yuanhong; Zhao, Chen; Jiang, Xuejun; Chen, Hongduo; Lang, Ming-Fei; Sun, Jing

    2014-01-01

    Cyclin A1 belongs to the type-A cyclins and participates in cell cycle regulation. Since its discovery, cyclin A1 has been shown mostly in testis. It plays important roles in spermatogenesis. However, there were also reports on ovary expression of cyclin A1. Therefore, we intended to revisit the expression of cyclin A1 in mouse ovary. Our study showed that cyclin A1 was expressed at the mRNA level and the protein level in mouse ovary. Tissue staining revealed that cyclin A1 was expressed in maturating oocytes. With the recent data on the functions of cyclins in somatic and stem cells, we also discussed the possibilities of further studies of cyclin A1 in mouse oocytes and perhaps in the oogonial stem cells. Our findings not only add to the supportive evidence of cyclin A1 expression in oocytes, but also may promote more interest in exploring cyclin A1 functions in ovary.

  3. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

  4. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    PubMed Central

    Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and

  5. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum.

    PubMed

    Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum's intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and

  6. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-10-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

  7. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed Central

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  8. Gene expression profile analysis of type 2 diabetic mouse liver.

    PubMed

    Zhang, Fang; Xu, Xiang; Zhang, Yi; Zhou, Ben; He, Zhishui; Zhai, Qiwei

    2013-01-01

    Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.

  9. CREBZF expression and hormonal regulation in the mouse uterus

    PubMed Central

    2013-01-01

    Background CREBZF is a member of the mammalian ATF/CREB family of the basic region-leucine zipper (bZIP) transcription factors. Two isoforms of CREBZF have been identified from the alternative usage of initiation codons, SMILE (long isoform of CREBZF) and Zhangfei (short isoform of CREBZF). Until recently, the physiological function of CREBZF in mammalian reproductions has not been reported. Methods Multiple techniques were performed to investigate the spatiotemporal expression and hormonal regulation of the CREBZF gene in the mouse uterus and its role in embryo implantation. Results Zhangfei was not detected in the mouse uterus. SMILE immunostaining was mainly expressed in the uterine luminal and glandular epithelium, and the expression levels of both SMILE mRNA and protein gradually decreased from days 1–3 of pregnancy, peaked on day 4, and then declined again on day 6. On day 5 of pregnancy, SMILE protein expression was detected only in the luminal epithelium at implantation sites compared with the expression at inter-implantation sites. SMILE protein was not detected in decidual cells from days 6–8 of pregnancy or artificial decidualisation. Furthermore, SMILE protein was not detected in the mouse uterus on days 3–6 of pseudopregnancy, and SMILE expression was also induced in the delayed-implantation uterus, indicating that the presence of an active blastocyst was required for SMILE expression at the implantation site. Oestrogen significantly stimulated SMILE expression in the ovariectomised mouse uterus. In addition, in cycling mice, high levels of SMILE protein and mRNA expression were also observed in proestrus and oestrus uteri. Conclusions Taken together, these results suggested that SMILE expression was closely related to mouse implantation and up-regulated by oestrogen. PMID:24325733

  10. The mouse Gene Expression Database (GXD): 2017 update

    PubMed Central

    Finger, Jacqueline H.; Smith, Constance M.; Hayamizu, Terry F.; McCright, Ingeborg J.; Xu, Jingxia; Law, Meiyee; Shaw, David R.; Baldarelli, Richard M.; Beal, Jon S.; Blodgett, Olin; Campbell, Jeff W.; Corbani, Lori E.; Lewis, Jill R.; Forthofer, Kim L.; Frost, Pete J.; Giannatto, Sharon C.; Hutchins, Lucie N.; Miers, Dave B.; Motenko, Howie; Stone, Kevin R.; Eppig, Janan T.; Kadin, James A.; Richardson, Joel E.; Ringwald, Martin

    2017-01-01

    The Gene Expression Database (GXD; www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. Through curation of the scientific literature and by collaborations with large-scale expression projects, GXD collects and integrates data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. Expression data from both wild-type and mutant mice are included. The expression data are combined with genetic and phenotypic data in Mouse Genome Informatics (MGI) and made readily accessible to many types of database searches. At present, GXD includes over 1.5 million expression results and more than 300 000 images, all annotated with detailed and standardized metadata. Since our last report in 2014, we have added a large amount of data, we have enhanced data and database infrastructure, and we have implemented many new search and display features. Interface enhancements include: a new Mouse Developmental Anatomy Browser; interactive tissue-by-developmental stage and tissue-by-gene matrix views; capabilities to filter and sort expression data summaries; a batch search utility; gene-based expression overviews; and links to expression data from other species. PMID:27899677

  11. Efficacious Cyclic N-Acyl O-Amino Phenol Duocarmycin Prodrugs

    PubMed Central

    Wolfe, Amanda L.; Duncan, Katharine K.; Parelkar, Nikhil K.; Brown, Douglas; Vielhauer, George A.; Boger, Dale L.

    2013-01-01

    Two novel cyclic N-acyl O-amino phenol prodrugs are reported as new members of a unique class of reductively cleaved prodrugs of the duocarmycin family of natural products. These prodrugs were explored with the expectation that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles and were designed to liberate the free drug without the release of an extraneous group. In vivo evaluation of the prodrug 6 showed that it exhibits extraordinary efficacy (T/C > 1500, L1210; 6/10 one year survivors) substantially exceeding that of the free drug, that its therapeutic window of activity is much larger permitting a dosing ≥ 40-fold higher than the free drug, and yet that it displays a potency in vivo that approaches the free drug (within 3-fold). Clearly, the prodrug 6 benefits from either its controlled slow release of the free drug or its preferential intracellular reductive cleavage. PMID:23627265

  12. N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens.

    PubMed

    Hernández-Reyes, Casandra; Schenk, Sebastian T; Neumann, Christina; Kogel, Karl-Heinz; Schikora, Adam

    2014-11-01

    The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens.

  13. Pseudomonas cremoricolorata Strain ND07 Produces N-acyl Homoserine Lactones as Quorum Sensing Molecules

    PubMed Central

    Yunos, Nina Yusrina Muhamad; Tan, Wen-Si; Koh, Chong-Lek; Sam, Choon-Kook; Mohamad, Nur Izzati; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Quorum sensing (QS) is a bacterial cell-to-cell communication system controlling QS-mediated genes which is synchronized with the population density. The regulation of specific gene activity is dependent on the signaling molecules produced, namely N-acyl homoserine lactones (AHLs). We report here the identification and characterization of AHLs produced by bacterial strain ND07 isolated from a Malaysian fresh water sample. Molecular identification showed that strain ND07 is clustered closely to Pseudomonas cremoricolorata. Spent culture supernatant extract of P. cremoricolorata strain ND07 activated the AHL biosensor Chromobacterium violaceum CV026. Using high resolution triple quadrupole liquid chromatography-mass spectrometry, it was confirmed that P. cremoricolorata strain ND07 produced N-octanoyl-l-homoserine lactone (C8-HSL) and N-decanoyl-l-homoserine lactone (C10-HSL). To the best of our knowledge, this is the first documentation on the production of C10-HSL in P. cremoricolorata strain ND07. PMID:24984061

  14. N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens

    PubMed Central

    Hernández-Reyes, Casandra; Schenk, Sebastian T; Neumann, Christina; Kogel, Karl-Heinz; Schikora, Adam

    2014-01-01

    The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens. PMID:25234390

  15. Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa.

    PubMed

    Hansen, Mette R; Jakobsen, Tim H; Bang, Claus G; Cohrt, Anders Emil; Hansen, Casper L; Clausen, Janie W; Le Quement, Sebastian T; Tolker-Nielsen, Tim; Givskov, Michael; Nielsen, Thomas E

    2015-04-01

    In an attempt to devise new antimicrobial treatments for biofilm infections, the bacterial cell-cell communication system termed quorum sensing has emerged as an attractive target. It has proven possible to intercept the communication system by synthetic non-native ligands and thereby lower the pathogenesis and antibiotic tolerance of a bacterial biofilm. To identify the structural elements important for antagonistic or agonistic activity against the Pseudomonas aeruginosa LasR protein, we report the synthesis and screening of new triazole-containing mimics of natural N-acyl homoserine lactones. A series of azide- and alkyne-containing homoserine lactone building blocks was used to prepare an expanded set of 123 homoserine lactone analogues through a combination of solution- and solid-phase synthesis methods. The resulting compounds were subjected to cell-based quorum sensing screening assays, thereby revealing several bioactive compounds, including 13 compounds with antagonistic activity and 9 compounds with agonistic activity.

  16. Pantoea sp. isolated from tropical fresh water exhibiting N-acyl homoserine lactone production.

    PubMed

    Tan, Wen-Si; Muhamad Yunos, Nina Yusrina; Tan, Pui-Wan; Mohamad, Nur Izzati; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.

  17. Novel acridine-based N-acyl-homoserine lactone analogs induce endoreduplication in the human oral squamous carcinoma cell line SAS.

    PubMed

    Chai, Hongbo; Hazawa, Masaharu; Hosokawa, Yoichiro; Igarashi, Jun; Suga, Hiroaki; Kashiwakura, Ikuo

    2012-01-01

    The cytotoxicity of novel acridine-based N-acyl-homoserine lactone (AHL) analogs was investigated on the human oral squamous carcinoma cell line SAS. One analog induced G2/M phase arrest at 5.3-10.6 µM and induced polyploidy at a higher dose (21.2 µM). Importantly, treatment of SAS cells with a combination of the AHL analog and the Jun N-terminal kinase (JNK) inhibitor, SP600125, prevented mitosis and induced polyploidy. The AHL analog synergized with X-irradiation to inhibit clonogenic survival of SAS cells; however, its radiosensitizing effects were relative to not X-irradiation-induced apoptosis but mitotic failure following enhanced expression of Aurora A and B. These results suggest that the active AHL analog showed growth-suppressive and radiosensitizing effects, which involve polyploidy followed by G2/M accumulation and atypical cell death in the SAS cell line.

  18. Differential expression and regulation of Tdo2 during mouse decidualization.

    PubMed

    Li, Dang-Dang; Gao, Ying-Jie; Tian, Xue-Chao; Yang, Zhan-Qing; Cao, Hang; Zhang, Qiao-Ling; Guo, Bin; Yue, Zhan-Peng

    2014-01-01

    Tryptophan 2,3-dioxygenase (Tdo2) is a rate-limiting enzyme which directs the conversion of tryptophan to kynurenine. The aim of this study was to examine the expression and regulation of Tdo2 in mouse uterus during decidualization. Tdo2 mRNA was mainly expressed in the decidua on days 6-8 of pregnancy. By real-time PCR, a high level of Tdo2 expression was observed in the uteri from days 6 to 8 of pregnancy, although Tdo2 expression was observed on days 1-8. Simultaneously, Tdo2 mRNA was also detected under in vivo and in vitro artificial decidualization. Estrogen, progesterone, and 8-bromoadenosine-cAMP could induce the expression of Tdo2 in the ovariectomized mouse uterus and uterine stromal cells. Tdo2 could regulate cell proliferation and stimulate the expression of decidual marker Dtprp in the uterine stromal cells and decidual cells. Overexpression of Tdo2 could upregulate the expression of Ahr, Cox2, and Vegf genes in uterine stromal cells, while Tdo2 inhibitor 680C91 could downregulate the expression of Cox2 and Vegf genes in uterine decidual cells. These data indicate that Tdo2 may play an important role during mouse decidualization and be regulated by estrogen, progesterone, and cAMP.

  19. MicroRNA expression in the aging mouse thymus.

    PubMed

    Ye, Yaqiong; Li, Daotong; Ouyang, Dan; Deng, Li; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2014-09-01

    MicroRNAs (miRNAs) have been implicated in the process of aging in many model organisms, such as Caenorhabditis elegans, and in many organs, such as the mouse lung and human epididymis. However, the role of miRNAs in the thymus tissues of the aging mouse remains unclear. To address this question, we investigated the miRNA expression profiles in the thymuses of 1-, 10- and 19-month-old mice using miRNA array and qRT-PCR assays. A total of 223 mouse miRNAs were screened, and the expression levels of those miRNAs exhibited gradual increases and decreases over the course of thymus aging. Fifty miRNAs in the 10-month-old thymus and 81 miRNAs in the 19-month-old thymus were defined as differentially expressed miRNAs (p<0.05) in comparison with their levels in the 1-month-old mouse, and approximately one-third of these miRNAs were grouped within 11 miRNA clusters. Each miRNA cluster contained 2 to 5 miRNA genes, and most of the cluster members displayed similar expression patterns, being either increased or decreased. In addition, Ingenuity Pathway Analysis (IPA) software and the IPA database were used to analyze the 12 miRNAs that exhibited significant expression changes, revealing that as many as 15 pathways may be involved. Thus, our current study determined the expression profiles of miRNAs in the mouse thymus during the process of aging. The results suggested that these miRNAs could become meaningful biomarkers for studying thymus aging and that the aging-related alternations in miRNA expression may be involved in the regulation of cell proliferation, apoptosis, development and carcinogenesis/tumorigenesis.

  20. Expression of HSG is essential for mouse blastocyst formation

    SciTech Connect

    Jiang Guangjian; Pan Lei; Huang Xiuying; Han Mei; Wen Jinkun . E-mail: wjk@hebmu.edu.cn; Sun Fangzhen . E-mail: fzsun@genetics.ac.cn

    2005-09-23

    It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with {beta}-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development.

  1. The mouse Gene Expression Database (GXD): 2014 update.

    PubMed

    Smith, Constance M; Finger, Jacqueline H; Hayamizu, Terry F; McCright, Ingeborg J; Xu, Jingxia; Berghout, Joanne; Campbell, Jeff; Corbani, Lori E; Forthofer, Kim L; Frost, Pete J; Miers, Dave; Shaw, David R; Stone, Kevin R; Eppig, Janan T; Kadin, James A; Richardson, Joel E; Ringwald, Martin

    2014-01-01

    The Gene Expression Database (GXD; http://www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. GXD collects different types of expression data from studies of wild-type and mutant mice, covering all developmental stages and including data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. The data are acquired from the scientific literature and from researchers, including groups doing large-scale expression studies. Integration with the other data in Mouse Genome Informatics (MGI) and interconnections with other databases places GXD's gene expression information in the larger biological and biomedical context. Since the last report, the utility of GXD has been greatly enhanced by the addition of new data and by the implementation of more powerful and versatile search and display features. Web interface enhancements include the capability to search for expression data for genes associated with specific phenotypes and/or human diseases; new, more interactive data summaries; easy downloading of data; direct searches of expression images via associated metadata; and new displays that combine image data and their associated annotations. At present, GXD includes >1.4 million expression results and 250,000 images that are accessible to our search tools.

  2. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  3. Reduced Expression of SARM in Mouse Spleen during Polymicrobial Sepsis.

    PubMed

    Gong, Yu; Zou, Lin; Cen, Dongzhi; Chao, Wei; Chen, Dunjin

    2016-12-01

    Objective Immune dysfunction, including prominent apoptosis of immune cells and decreased functioning of the remaining immune cells, plays a central role in the pathogenesis of sepsis. Sterile α and HEAT/armadillo motif-containing protein (SARM) is implicated in the regulation of immune cell apoptosis. This study aimed to elucidate SARM contributes to sepsis-induced immune cell death and immunosuppression. Methods A mouse model of polymicrobial sepsis was generated by cecum ligation and puncture (CLP). SARM gene and protein expression, caspase 3 cleavage and intracellular ATP production were measured in the mouse spleens. Results CLP-induced polymicrobial sepsis specifically attenuated both the gene and protein expression of SARM in the spleens. Moreover, the attenuation of SARM expression synchronized with splenocyte apoptosis, as evidenced by increased caspase 3 cleavage and ATP depletion. Conclusions These findings suggest that SARM is a potential regulator of sepsis-induced splenocyte apoptosis.

  4. EMAGE mouse embryo spatial gene expression database: 2014 update

    PubMed Central

    Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Moss, Julie; Graham, Liz; Burton, Nicholas; Hill, Bill; Rao, Jianguo; Baldock, Richard A.; Armit, Chris

    2014-01-01

    EMAGE (http://www.emouseatlas.org/emage/) is a freely available database of in situ gene expression patterns that allows users to perform online queries of mouse developmental gene expression. EMAGE is unique in providing both text-based descriptions of gene expression plus spatial maps of gene expression patterns. This mapping allows spatial queries to be accomplished alongside more traditional text-based queries. Here, we describe our recent progress in spatial mapping and data integration. EMAGE has developed a method of spatially mapping 3D embryo images captured using optical projection tomography, and through the use of an IIP3D viewer allows users to view arbitrary sections of raw and mapped 3D image data in the context of a web browser. EMAGE now includes enhancer data, and we have spatially mapped images from a comprehensive screen of transgenic reporter mice that detail the expression of mouse non-coding genomic DNA fragments with enhancer activity. We have integrated the eMouseAtlas anatomical atlas and the EMAGE database so that a user of the atlas can query the EMAGE database easily. In addition, we have extended the atlas framework to enable EMAGE to spatially cross-index EMBRYS whole mount in situ hybridization data. We additionally report on recent developments to the EMAGE web interface, including new query and analysis capabilities. PMID:24265223

  5. Expression and Function of Kisspeptin during Mouse Decidualization

    PubMed Central

    Lin, Yan; Zong, Teng; Zhong, Chengxue; Zhang, BaoPing; Ren, Min; Kuang, HaiBin

    2014-01-01

    Background Plasma kisspeptin levels dramatically increased during the first trimester of human pregnancy, which is similar to pregnancy specific glycoprotein-human chorionic gonadotropin. However, its particular role in the implantation and decidualization has not been fully unraveled. Here, the study was conducted to investigate the expression and function of kisspeptin in mouse uterus during early pregnancy and decidualization. Methodology/Principal Findings Quantitative PCR results demonstrated that Kiss1 and GPR54 mRNA levels showed dynamic increase in the mouse uterus during early pregnancy and artificially induced decidualization in vivo. KISS-1 and GPR54 proteins were spatiotemporally expressed in decidualizing stromal cells in intact pregnant females, as well as in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Kiss1 mRNA was upregulated after progesterone or/and estradiol treatment. Moreover, in a stromal cell culture model, the expression of Kiss1 and GPR54 mRNA gradually rise with the progression of stromal cell decidualization, whereas the attenuated expression of Kiss1 using small interfering RNA approaches significantly blocked the progression of stromal cell decidualization. Conclusion our results demonstrated that Kiss1/GPR54 system was involved in promoting uterine decidualization during early pregnancy in mice. PMID:24830702

  6. Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

    PubMed

    Yamada, Takahiro; Takemura, Yoshizumi; Niisato, Naomi; Mitsuyama, Etsuko; Iwasaki, Yoshinobu; Marunaka, Yoshinori

    2009-03-13

    We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

  7. Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

    PubMed

    Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

    2008-01-01

    The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

  8. Expression profiling of the mouse early embryo: Reflections and Perspectives

    PubMed Central

    Ko, Minoru S. H.

    2008-01-01

    Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

  9. Progesterone regulates secretin expression in mouse uterus during early pregnancy.

    PubMed

    Huang, Zhu; Wang, Tong-Song; Qi, Qian-Rong; Zuo, Ru-Juan; Liang, Xiao-Huan; Zhao, Xu-Yu; Yang, Zeng-Ming

    2014-06-01

    Secretin, a classical gastrointestinal and neuroendocrine peptide, plays an important role in maintaining the body fluid balance. However, the expression and regulation of secretin in the reproductive system are still unknown. In our study, secretin is specifically expressed in the decidua on days 5 to 8 of pregnancy. Secretin expression is not detected under delayed implantation but is stimulated after estrogen activation and under artificial decidualization. Progesterone induces secretin expression in ovariectomized mice and cultured stromal cells, which is abrogated by specific LY294002. Because secretin is mainly localized in the decidua and also strongly expressed during in vitro decidualization, secretin may play a role during mouse decidualization through regulating cyclic adenosine monophosphate level.

  10. Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

    PubMed

    Budunova, I V; Carbajal, S; Kang, H; Viaje, A; Slaga, T J

    1997-03-01

    Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

  11. MomL, a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

    PubMed Central

    Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom

    2014-01-01

    Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105 s−1 M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. PMID:25398866

  12. MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia.

    PubMed

    Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom; Zhang, Xiao-Hua

    2015-01-01

    Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 10(5) s(-1) M(-1). Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the "HXHXDH" motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.

  13. Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.

    PubMed

    Sio, Charles F; Otten, Linda G; Cool, Robbert H; Diggle, Stephen P; Braun, Peter G; Bos, Rein; Daykin, Mavis; Cámara, Miguel; Williams, Paul; Quax, Wim J

    2006-03-01

    The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

  14. Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

    PubMed Central

    Sio, Charles F.; Otten, Linda G.; Cool, Robbert H.; Diggle, Stephen P.; Braun, Peter G.; Bos, Rein; Daykin, Mavis; Cámara, Miguel; Williams, Paul; Quax, Wim J.

    2006-01-01

    The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies. PMID:16495538

  15. Expression of luteinizing hormone receptors in the mouse penis.

    PubMed

    Kokk, Kersti; Kuuslahti, Marianne; Keisala, Tiina; Purmonen, Sami; Kaipia, Antti; Tammela, Teuvo; Orro, Helen; Simovart, Helle-Evi; Pöllänen, Pasi

    2011-01-01

    The role of luteinizing hormone (LH) in the regulation of normal reproductive functions in males and females is quite well established. Besides the expression of LH receptors in the target cells in gonads, it has been found in several extragonadal organs. There is no information about the expression of LH receptors in the penis up to now. The aim of the present study is to investigate the expression of the LH receptor in the mouse penis to see if LH effects are possible in the penis. BALB/c mice were used as donors of normal penis and testis tissue. Immunocytochemistry, Western blotting, and quantitative reverse transcriptase polymerase chain reactions (RT-PCRs) were used for the detection of the LH receptor. Positive immunoreaction for LH receptors was present in the nuclei of urethral epithelium and endothelial cells of cavernous spaces in the corpus cavernosum and corpus spongiosum penis. Western blotting experiments demonstrated the presence of LH antigen at M(r) = 97.4 and 78 kd. Quantitative RT-PCRs confirmed the expression of LH receptor in the penis. Our results show that LH receptor is expressed in the body of the mouse penis; thus, it may directly regulate functions of penile tissue.

  16. Gene expression and dental enamel structure in developing mouse incisor.

    PubMed

    Sehic, Amer; Risnes, Steinar; Khan, Qalb-E-Saleem; Khuu, Cuong; Osmundsen, Harald

    2010-04-01

    At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

  17. 3D confocal reconstruction of gene expression in mouse.

    PubMed

    Hecksher-Sørensen, J; Sharpe, J

    2001-01-01

    Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

  18. PRDM16 expression in the developing mouse embryo.

    PubMed

    Horn, Kristin H; Warner, Dennis R; Pisano, Michele; Greene, Robert M

    2011-02-01

    PRDM16 is a member of the PR domain-containing protein family and is associated with various disease states including myelodysplastic syndrome and adult T-cell leukemia, as well as developmental abnormalities such as cleft palate. It is also known to act as a regulator of cell differentiation. Expression analysis of PRDM16 is limited, especially within the developing embryo. The current study evaluated the temporal and spatial localization of PRDM16 during early mouse development (embryonic days 8.5-14.5). PRDM16 was first detected on E9.5 in a limited number of tissues and by E14.5, was expressed in a broad range of developing tissues including those of the brain, lung, kidney, and gastrointestinal tract. The expression pattern is consistent with a role for PRDM16 in the development of multiple tissues. Collectively, these studies are the first to characterize the expression of the PRDM16 gene during early murine development.

  19. Sex-specific gene expression in the BXD mouse liver.

    PubMed

    Gatti, Daniel M; Zhao, Ni; Chesler, Elissa J; Bradford, Blair U; Shabalin, Andrey A; Yordanova, Roumyana; Lu, Lu; Rusyn, Ivan

    2010-08-01

    Differences in clinical phenotypes between the sexes are well documented and have their roots in differential gene expression. While sex has a major effect on gene expression, transcription is also influenced by complex interactions between individual genetic variation and environmental stimuli. In this study, we sought to understand how genetic variation affects sex-related differences in liver gene expression by performing genetic mapping of genomewide liver mRNA expression data in a genetically defined population of naive male and female mice from C57BL/6J, DBA/2J, B6D2F1, and 37 C57BL/6J x DBA/2J (BXD) recombinant inbred strains. As expected, we found that many genes important to xenobiotic metabolism and other important pathways exhibit sexually dimorphic expression. We also performed gene expression quantitative trait locus mapping in this panel and report that the most significant loci that appear to regulate a larger number of genes than expected by chance are largely sex independent. Importantly, we found that the degree of correlation within gene expression networks differs substantially between the sexes. Finally, we compare our results to a recently released human liver gene expression data set and report on important similarities in sexually dimorphic liver gene expression between mouse and human. This study enhances our understanding of sex differences at the genome level and between species, as well as increasing our knowledge of the molecular underpinnings of sex differences in responses to xenobiotics.

  20. EMAGE mouse embryo spatial gene expression database: 2010 update

    PubMed Central

    Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A.; Davidson, Duncan R.; Christiansen, Jeffrey H.

    2010-01-01

    EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (∼19 000 gene) ‘EURExpress’ dataset into EMAGE. PMID:19767607

  1. The expression of SEIPIN in the mouse central nervous system.

    PubMed

    Liu, Xiaoyun; Xie, Beibei; Qi, Yanfei; Du, Ximing; Wang, Shaoshi; Zhang, Yumei; Paxinos, George; Yang, Hongyuan; Liang, Huazheng

    2016-11-01

    Immunohistochemical staining was used to investigate the expression pattern of SEIPIN in the mouse central nervous system. SEIPIN was found to be present in a large number of areas, including the motor and somatosensory cortex, the thalamic nuclei, the hypothalamic nuclei, the mesencephalic nuclei, some cranial motor nuclei, the reticular formation of the brainstem, and the vestibular complex. Double labeling with NeuN antibody confirmed that SEIPIN-positive cells in some nuclei were neurons. Retrograde tracer injections into the spinal cord revealed that SEIPIN-positive neurons in the motor and somatosensory cortex and other movement related nuclei project to the mouse spinal cord. The present study found more nuclei positive for SEIPIN than shown using in situ hybridization and confirmed the presence of SEIPIN in neurons projecting to the spinal cord. The results of this study help to explain the clinical manifestations of patients with Berardinelli-Seip congenital lipodystrophy (Bscl2) gene mutations.

  2. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  3. Expression of DMP1 in the developing mouse tongue embryo.

    PubMed

    Murata, Hidetaka; Sunohara, Msataka; Sato, Iwao

    2015-07-01

    Dentin matrix protein 1 (DMP-1) is an important factor in the mineralization of hard tissues. However, it has many other functions in addition to the regulation of mineralized tissues. We analyzed the expression and localization of DMP-1 by immunohistochemical staining and in situ hybridization in the developing mouse tongue during embryonic days 12.5 (E12.5), E14.5, E17.5, and E18.5. We also detected the mRNA abundance of tongue morphogenesis markers such as FGF6, TGF-β1, Collagen I, osteocalcin, chondromodulin 1, tenomodulin, Vascular endothelial growth factor (VEGF), caspase-3, and Aifm from embryonic stages by real-time RT-PCR. The antisense probe for DMP-1 was detected in a few mesenchymal cells surrounding blood vessels at E12.5, and faint localization was seen at E18.5 in the embryonic mouse tongue by in situ hybridization. The DMP-1 and osteocalcin abundance levels gradually increased compared with the other tongue markers from E12.5 to E18.5 (p<0.001). Cluster analyses identified the following distinct clusters for mRNA abundance in the tongue: cluster 1, E12.5; cluster 2, E14.5 and E17.5; and cluster 3, E18.5. The positive correlation between DMP-1 and osteocalcin (Pearson's r=0.685; p<0.05) and negative correlation between DMP-1 and Caspase-3 (Pearson's r=-0.632; p<0.05) were analyzed. These data suggested that DMP-1 potentially influences osteocalcin and Caspase-3 during mouse tongue development and morphogenesis. DMP-1 also affects the angiogenic marker VEGF in specific stages and areas, terminating the differentiation of the tongue from other developing tissues. We conclude that DMP-1 may be involved in regulating the temporal expression at embryonic stages in the mouse tongue.

  4. Developmental expression profiles of Celsr (Flamingo) genes in the mouse.

    PubMed

    Tissir, F; De-Backer, O; Goffinet, A M; Lambert de Rouvroit, C

    2002-03-01

    Celsr, also called Flamingo (Fmi) genes encode proteins of the cadherin superfamily. Celsr cadherins are seven-pass transmembrane proteins with nine cadherin repeats in the extracellular domain, and an anonymous intracellular C-terminus. The Drosophila Fmi gene regulates epithelial planar cell polarity and dendritic field deployment. The three Flamingo gene orthologs in man and rodents are named, respectively, CELSR1-3 and Celsr1-3. Celsr1 and 2 are expressed during early development, in the brain and epithelia. In this report, we characterized further Celsr genes in the mouse, and examined their developmental pattern of expression. Each Celsr is expressed prominently in the developing brain following a specific pattern, suggesting that they serve distinct functions.

  5. Gene expression profiling of mouse embryos with microarrays

    PubMed Central

    Sharov, Alexei A.; Piao, Yulan; Ko, Minoru S. H.

    2011-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. PMID:20699157

  6. Expression of liver alpha-amylase in obese mouse hepatocytes

    PubMed Central

    Afsartala, Zohreh; Savabkar, Sanaz; Nazemalhosseini Mojarad, Ehsan; Assadollahi, Vahideh; Tanha, Shima; Bijangi, Khosro; Gholami, Mohammadreza

    2016-01-01

    Aim: The aim of this study is to demonstrate the relation between the expression of liver alpha-amylase and obesity. Background: Alpha-amylase catalyses the hydrolysis of 1, 4-alpha-glucosidic linkages in polysaccharides and has three main subtypes, including: salivary, pancreatic, and hepatic. Hepatic alpha-amylase is involved in glycogen metabolism, and has a role in obesity and its management. In this study, we aimed to analyze the expression of liver alpha-amylase in overweight and obese mouse. Material and methods: In this study, NMRI male mice were randomly divided into two groups. The sample group (obese) took a high-fat and carbohydrate diet, while the control group (normal) took a laboratory pellet chow for eight weeks. During this period, their weight was measured. After eight weeks, liver hepatocytes were isolated using an enzymatic digestion method. Immunocytochemistry (ICC) and flow cytometry analysis were performed to measure alpha amylase protein expression in mouse liver hepatocyte cells. Results: A significant difference in the body weight was observed between the two groups (p<0.05). The qualitative protein expression of liver alpha-amylase was found to be higher in the obese group in both tests (immunocytochemistry and flow cytometry). Animals from the test group presented higher alpha-amylase expression, which suggests that this hepatic protein may constitute a potential indicator of susceptibility for fat tissue accumulation and obesity. The present data demonstrates an increased expression of liver amylase in obese mice. Conclusion: These results suggest that liver amylase secretion might be useful for predicting susceptibility to obesity induced by consumption of a high-fat and carbohydrate diet. PMID:27895853

  7. Differential extra-renal expression of the mouse renin genes.

    PubMed Central

    Miller, C C; Carter, A T; Brooks, J I; Lovell-Badge, R H; Brammar, W J

    1989-01-01

    We have used RNase-protection analyses to study renin gene expression in one- and two-gene mouse strains. The RNase-protection assay is capable of discriminating between the transcripts from the different renin genes. In a two-gene strain containing Ren-1D and Ren-2, we demonstrate transcriptional activity from Ren-1D in kidney, submandibular gland (SMG), testes, liver, brain and heart. Ren-2 is clearly expressed in kidney, SMG and testes. Similar analyses of one gene strains (containing Ren-1C only) show expression in kidney, SMG, testes, brain and heart. We cannot detect renin mRNA in the liver of these mice. Ren-1C and Ren-1D thus display quite different tissue-specificities. In order to determine whether the different tissue-specificities of the highly homologous Ren-1C and Ren-1D genes are due to different trans-acting factors in the different mouse strains or to different cis-acting DNA elements inherent to the genes, we introduced a Ren-1D transgene (Ren-1*) into a background strain containing only the Ren-1C gene. The transgene exhibits the same tissue-specificity as the Ren-1D gene of two-gene strains suggesting the presence of different cis-acting DNA elements in Ren-1C and Ren-1D. Images PMID:2657654

  8. A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups.

    PubMed

    Kristová, Veronika; Martínková, Ludmila; Husáková, Lucie; Kuzma, Marek; Rauvolfová, Jana; Kavan, Daniel; Pompach, Petr; Bezouska, Karel; Kren, Vladimír

    2005-01-26

    The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.

  9. Inheritance and expression of the mouse metallothionein gene in tobacco

    SciTech Connect

    Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. )

    1989-11-01

    Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.

  10. Identification and characterization of a novel N-acyl-homoserine lactonase gene in Sphingomonas ursincola isolated from industrial cooling water systems.

    PubMed

    Morohoshi, Tomohiro; Sato, Niina; Iizumi, Taro; Tanaka, Airi; Ikeda, Tsukasa

    2017-01-24

    Biofilm formation by bacteria is one of the main causes of fouling in industrial cooling water systems. In many gram-negative bacteria, biofilm formation is regulated by N-acyl-homoserine lactone (AHL)-mediated quorum sensing. In this study, we isolated three AHL-degrading bacteria from cooling water systems and identified them as Sphingomonas ursincola. The draft genome sequence of S. ursincola A1 revealed the presence of an AHL-degrading gene homolog, designated qsdS. The qsdS region was also amplified by PCR from the genomes of the other two S. ursincola strains, SF1 and SF8. Escherichia coli DH5α harboring a QsdS-expressing plasmid showed high degradative activity against AHLs with short and 3-oxo-substituted acyl chains. High-performance liquid chromatography analysis revealed that QsdS is an AHL lactonase, an enzyme that catalyzes AHL ring opening. Furthermore, heterologous expression of QsdS in Pseudomonas aeruginosa PAO1 resulted in degradation of endogenous AHLs and interfered with the quorum-sensing-regulated phenotype.

  11. Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

    PubMed

    Lin, Pei-Hsun; Su, Shiun-Cheng; Tsai, Ying-Chieh; Lee, Chia-Yin

    2002-10-01

    An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.

  12. Expression of FGFs during early mouse tongue development

    PubMed Central

    Du, Wen; Prochazka, Jan; Prochazkova, Michaela; Klein, Ophir D.

    2016-01-01

    The fibroblast growth factors (FGFs) constitute one of the largest growth factor families, and several ligands and receptors in this family are known to play critical roles during tongue development. In order to provide a comprehensive foundation for research into the role of FGFs during the process of tongue formation, we measured the transcript levels by quantitative PCR and mapped the expression patterns by in situ hybridization of all 22 Fgfs during mouse tongue development between embryonic days (E) 11.5 and E14.5. During this period, Fgf5, Fgf6, Fgf7, Fgf9, Fgf10, Fgf13, Fgf15, Fgf16 and Fgf18 could all be detected with various intensities in the mesenchyme, whereas Fgf1 and Fgf2 were expressed in both the epithelium and the mesenchyme. Our results indicate that FGF signaling regulates tongue development at multiple stages. PMID:26748348

  13. Mouse ornithine decarboxylase gene: cloning, structure, and expression.

    PubMed Central

    Brabant, M; McConlogue, L; van Daalen Wetters, T; Coffino, P

    1988-01-01

    We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression. Images PMID:3353375

  14. TRP channel gene expression in the mouse retina.

    PubMed

    Gilliam, Jared C; Wensel, Theodore G

    2011-12-08

    In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium.

  15. Aquaporin-11 (AQP11) Expression in the Mouse Brain

    PubMed Central

    Koike, Shin; Tanaka, Yasuko; Matsuzaki, Toshiyuki; Morishita, Yoshiyuki; Ishibashi, Kenichi

    2016-01-01

    Aquaporin-11 (AQP11) is an intracellular aquaporin expressed in various tissues, including brain tissues in mammals. While AQP11-deficient mice have developed fatal polycystic kidneys at one month old, the role of AQP11 in the brain was not well appreciated. In this study, we examined the AQP11 expression in the mouse brain and the brain phenotype of AQP11-deficient mice. AQP11 messenger ribonucleic acid (mRNA) and protein were expressed in the brain, but much less than in the thymus and kidney. Immunostaining showed that AQP11 was localized at the epithelium of the choroid plexus and at the endothelium of the brain capillary, suggesting that AQP11 may be involved in water transport at the choroid plexus and blood-brain barrier (BBB) in the brain. The expression of AQP4, another brain AQP expressed at the BBB, was decreased by half in AQP11-deficient mice, thereby suggesting the presence of the interaction between AQP11 and AQP4. The brain of AQP11-deficient mice, however, did not show any morphological abnormalities and the function of the BBB was intact. Our findings provide a novel insight into a water transport mechanism mediated by AQPs in the brain, which may lead to a new therapy for brain edema. PMID:27258268

  16. Gene expression profiling of the developing mouse kidney and embryo.

    PubMed

    Shaw, Lisa; Johnson, Penny A; Kimber, Susan J

    2010-02-01

    The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.

  17. Lipid bilayers containing sphingomyelins and ceramides of varying N-acyl lengths: a glimpse into sphingolipid complexity.

    PubMed

    Jiménez-Rojo, Noemi; García-Arribas, Aritz B; Sot, Jesús; Alonso, Alicia; Goñi, Félix M

    2014-01-01

    The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain.

  18. Altered gene expression profiles in mouse tetraploid blastocysts.

    PubMed

    Park, Mi-Ryung; Hwang, Kyu-Chan; Bui, Hong-Thuy; Cho, Ssang-Goo; Park, Chankyu; Song, Hyuk; Oh, Jae-Wook; Kim, Jin-Hoi

    2012-01-01

    In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (P<0.05). Of these 50 genes, 28 were more highly expressed in tetraploid-derived blastocysts, whereas 22 were more highly downregulated. However, some genes involved in receptor activity, cell adhesion molecule, calcium ion binding, protein biosynthesis, redox processes, transport, and transcription showed a significant decrease or increase in gene expression in the tetraploid-derived blastocyst embryos. Thus, microarray analysis can be used as a tool to screen for underlying defects responsible for the development of tetraploid-derived embryos.

  19. Aquaporin7 expression during perinatal development of mouse brain.

    PubMed

    Shin, Incheol; Kim, Hyun J; Lee, Jae E; Gye, Myung C

    2006-12-01

    Emerging evidence suggests that brain aquaporins (AQPs) play important roles in the dynamic regulation of brain water homeostasis and the production of cerebrospinal fluid (CSF) under normal, as well as pathological, conditions. To date, the spatiotemporal expression patterns of AQP1, 4, and 9 have been elucidated in brain tissues. However, the expression of AQP7, an aquaglyceroporin associated with brain development, has not been shown. In the present study, we examined expression of AQP7 during perinatal and adult brain development in the mouse. Throughout brain development, the immunoreactivity of AQP7 was largely found in the choroid plexus (CP). AQP7 immunoreactivity in ependyma (Ep), pia, and blood vessels (BV) was increased during perinatal to postnatal development. Cells in the different layers of cerebral cortex became a little positive for AQP7 immunoreactivity during postnatal development. Optimized semi-quantitative RT-PCR and Western blot analysis revealed that AQP7 mRNA and protein levels increased during perinatal development of brain. To our knowledge, this is the first report on the pattern of AQP7 expression in brain tissues. These results suggest that AQP7 is an important structural element in the choroid plexus and is possibly involved in the production of CSF during brain development in mice.

  20. Expression of cloned immunoglobulin genes introduced into mouse L cells.

    PubMed Central

    Gilles, S D; Tonegawa, S

    1983-01-01

    Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells. Images PMID:6316279

  1. A gene expression fingerprint of mouse stomach ECL cells.

    PubMed

    Andersson, Niklas; Skrtic, Sofia Movérare; Håkanson, Rolf; Ohlsson, Claes

    2005-07-01

    Many of the endocrine cells in the stomach are poorly characterized with respect to physiological significance. In some cases, the anticipated hormone has not yet been identified. Global gene expression analysis of mouse stomach was performed in an attempt to identify the ECL-cell peptide/protein. Specific functional activation (omeprazole-induced hypergastrinaemia) was used as a tool to generate a gene expression fingerprint of the ECL cells. The proposed fingerprint includes 14 genes, among them six are known to be expressed by ECL cells (=positive controls), and some novel ones, which are likely to be ECL-cell-related. The known ECL-cell-related genes are those encoding histidine decarboxylase, chromogranin A and B, vesicular monoamine transporter 2, synaptophysin, and the cholecystokinin-B receptor. In addition, the fingerprint included five genes, which might be involved in the process of secretion and three ESTs with unknown function. Interestingly, parathyroid hormone-like hormone (Pthlh) was identified as a candidate ECL-cell peptide hormone.

  2. SSAO/VAP-1 protein expression during mouse embryonic development.

    PubMed

    Valente, Tony; Solé, Montse; Unzeta, Mercedes

    2008-09-01

    SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation.

  3. c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation*

    PubMed Central

    Ishibashi, Riki; Kozuki, Satoshi; Kamakura, Sachiko; Sumimoto, Hideki; Toyoshima, Fumiko

    2016-01-01

    Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation. PMID:26694615

  4. c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Ishibashi, Riki; Kozuki, Satoshi; Kamakura, Sachiko; Sumimoto, Hideki; Toyoshima, Fumiko

    2016-02-12

    Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

  5. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    PubMed

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  6. Asymmetric Synthesis of a CBI-Based Cyclic N-Acyl O-Amino Phenol Duocarmycin Prodrug

    PubMed Central

    2015-01-01

    A short, asymmetric synthesis of a cyclic N-acyl O-amino phenol duocarmycin prodrug subject to reductive activation based on the simplified 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) DNA alkylation subunit is described. A key element of the approach entailed treatment of iodo-epoxide 7, prepared by N-alkylation of 6 with (S)-glycidal 3-nosylate, with EtMgBr at room temperature to directly provide the optically pure alcohol 8 in 78% yield (99% ee) derived from an effective metal–halogen exchange and subsequent regioselective intramolecular 6-endo-tet cyclization. Following O-debenzylation, introduction of a protected N-methylhydroxamic acid, direct trannannular spirocyclization, and subsequent stereoelectronically controlled acid-catalyzed cleavage of the resulting cyclopropane (HCl), further improvements in a unique intramolecular cyclization with N–O bond formation originally introduced for formation of the reductively labile prodrug functionality are detailed. PMID:25247380

  7. Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

    PubMed

    Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

    2010-11-15

    Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication.

  8. Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family

    PubMed Central

    Gan, Han Ming; Dailey, Lucas K.; Halliday, Nigel; Williams, Paul; Hudson, André O.

    2016-01-01

    Background Members of the genus Novosphingobium have been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated. Methods In this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by the N. subterraneum DSM 12447 strain. Results Three predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference. Discussion This study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae. PMID:27635318

  9. Expression of cubilin in mouse testes and Leydig cells.

    PubMed

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

  10. Expression profile and transcription factor binding site exploration of imprinted genes in human and mouse

    PubMed Central

    Steinhoff, Christine; Paulsen, Martina; Kielbasa, Szymon; Walter, Jörn; Vingron, Martin

    2009-01-01

    Background In mammals, imprinted genes are regulated by an epigenetic mechanism that results in parental origin-specific expression. Though allele-specific regulation of imprinted genes has been studied for several individual genes in detail, little is known about their overall tissue-specific expression patterns and interspecies conservation of expression. Results We performed a computational analysis of microarray expression data of imprinted genes in human and mouse placentae and in a variety of adult tissues. For mouse, early embryonic stages were also included. The analysis reveals that imprinted genes are expressed in a broad spectrum of tissues for both species. Overall, the relative tissue-specific expression levels of orthologous imprinted genes in human and mouse are not highly correlated. However, in both species distinctive expression profiles are found in tissues of the endocrine pathways such as adrenal gland, pituitary, pancreas as well as placenta. In mouse, the placental and embryonic expression patterns of imprinted genes are highly similar. Transcription factor binding site (TFBS) prediction reveals correlation of tissue-specific expression patterns and the presence of distinct TFBS signatures in the upstream region of human imprinted genes. Conclusion Imprinted genes are broadly expressed pre- and postnatally and do not exhibit a distinct overall expression pattern when compared to non-imprinted genes. The relative expression of most orthologous gene pairs varies significantly between human and mouse suggesting rapid species-specific changes in gene regulation. Distinct expression profiles of imprinted genes are confined to certain human and mouse hormone producing tissues, and placentae. In contrast to the overall variability, distinct expression profiles and enriched TFBS signatures are found in human and mouse endocrine tissues and placentae. This points towards an important role played by imprinted gene regulation in these tissues. PMID

  11. N-Acyl-Homoserine Lactone Confers Resistance toward Biotrophic and Hemibiotrophic Pathogens via Altered Activation of AtMPK61[C][W

    PubMed Central

    Schikora, Adam; Schenk, Sebastian T.; Stein, Elke; Molitor, Alexandra; Zuccaro, Alga; Kogel, Karl-Heinz

    2011-01-01

    Pathogenic and symbiotic bacteria rely on quorum sensing to coordinate the collective behavior during the interactions with their eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signals in such communication. Here we show that plants have evolved means to perceive AHLs and that the length of acyl moiety and the functional group at the γ position specify the plant’s response. Root treatment with the N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) reinforced the systemic resistance to the obligate biotrophic fungi Golovinomyces orontii in Arabidopsis (Arabidopsis thaliana) and Blumeria graminis f. sp. hordei in barley (Hordeum vulgare) plants. In addition, oxo-C14-HSL-treated Arabidopsis plants were more resistant toward the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000. Oxo-C14-HSL promoted a stronger activation of mitogen-activated protein kinases AtMPK3 and AtMPK6 when challenged with flg22, followed by a higher expression of the defense-related transcription factors WRKY22 and WRKY29, as well as the PATHOGENESIS-RELATED1 gene. In contrast to wild-type Arabidopsis and mpk3 mutant, the mpk6 mutant is compromised in the AHL effect, suggesting that AtMPK6 is required for AHL-induced resistance. Results of this study show that AHLs commonly produced in the rhizosphere are crucial factors in plant pathology and could be an agronomic issue whose full impact has to be elucidated in future analyses. PMID:21940998

  12. Identification of a set of genes showing regionally enriched expression in the mouse brain

    PubMed Central

    D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM

    2008-01-01

    Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. PMID:18625066

  13. Properties of recombinant mouse thrombospondin 2 expressed in Spodoptera cells.

    PubMed

    Chen, H; Sottile, J; O'Rourke, K M; Dixit, V M; Mosher, D F

    1994-12-23

    A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.

  14. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    PubMed

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  15. The gene expression database for mouse development (GXD): putting developmental expression information at your fingertips.

    PubMed

    Smith, Constance M; Finger, Jacqueline H; Kadin, James A; Richardson, Joel E; Ringwald, Martin

    2014-10-01

    Because molecular mechanisms of development are extraordinarily complex, the understanding of these processes requires the integration of pertinent research data. Using the Gene Expression Database for Mouse Development (GXD) as an example, we illustrate the progress made toward this goal, and discuss relevant issues that apply to developmental databases and developmental research in general. Since its first release in 1998, GXD has served the scientific community by integrating multiple types of expression data from publications and electronic submissions and by making these data freely and widely available. Focusing on endogenous gene expression in wild-type and mutant mice and covering data from RNA in situ hybridization, in situ reporter (knock-in), immunohistochemistry, reverse transcriptase-polymerase chain reaction, Northern blot, and Western blot experiments, the database has grown tremendously over the years in terms of data content and search utilities. Currently, GXD includes over 1.4 million annotated expression results and over 260,000 images. All these data and images are readily accessible to many types of database searches. Here we describe the data and search tools of GXD; explain how to use the database most effectively; discuss how we acquire, curate, and integrate developmental expression information; and describe how the research community can help in this process.

  16. [Synthesis of signaling N-acyl-homoserine-lactones participating in quorum sensing in rhizosphere and soil bacteria Pseudomonas and Xanthomonas].

    PubMed

    Khmel', I A; Veselova, M A; Metlitskaia, A Z; Klein, S; Lipasova, V A; Maiatskaia, A V; Chernin, L S

    2002-04-01

    Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonas and Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize this compounds. In at least a half of the isolated P. aureofaciens and P. aeruginosa, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri, P. geniculata, and P. putida. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.

  17. Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

    PubMed

    Nieto-Peñalver, Carlos G; Bertini, Elisa V; de Figueroa, Lucía I C

    2012-07-01

    The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

  18. Metabolomic Profiling Reveals the N-Acyl-Taurine Geodiataurine in Extracts from the Marine Sponge Geodia macandrewii (Bowerbank).

    PubMed

    Olsen, Elisabeth K; Søderholm, Kine L; Isaksson, Johan; Andersen, Jeanette H; Hansen, Espen

    2016-05-27

    A metabolomic approach was used to identify known and new natural products from the marine sponges Geodia baretti and G. macandrewii. G. baretti is known to produce bioactive natural products such as barettin (1), 8,9-dihydrobarettin (2), and bromobenzisoxazolone barettin (3), while secondary metabolites from G. macandrewii are not reported in the literature. Specimens of the two sponges were collected from different sites along the coast of Norway, and their extracts were analyzed using UHPLC-HR-MS. Metabolomic analyses revealed that extracts from both species contained barettin (1) and 8,9-dihydrobarettin (2), and all samples of G. baretti contained higher amounts of both compounds compared to G. macandrewii. The analysis of the MS data also revealed that samples of G. macandrewii contained a compound that was not present in any of the G. baretti samples. This new compound was isolated and identified as the N-acyl-taurine geodiataurine (4), and it was tested for antioxidant, anticancer, and antibacterial properties.

  19. Three-dimensional structure of the quorum-quenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis

    PubMed Central

    Liu, Dali; Lepore, Bryan W.; Petsko, Gregory A.; Thomas, Pei W.; Stone, Everett M.; Fast, Walter; Ringe, Dagmar

    2005-01-01

    The three-dimensional structure of the N-acyl-l-homoserine lactone hydrolase (AHL lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-Å resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL lactonases. These enzymes are members of the metallo-β-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals. PMID:16087890

  20. The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase.

    PubMed

    Kim, Myung Hee; Choi, Won-Chan; Kang, Hye Ok; Lee, Jong Suk; Kang, Beom Sik; Kim, Kyung-Jin; Derewenda, Zygmunt S; Oh, Tae-Kwang; Lee, Choong Hwan; Lee, Jung-Kee

    2005-12-06

    In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-angstroms resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-angstroms crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-beta-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases.

  1. Synthesis and Biological Evaluation of Triazole-Containing N-Acyl Homoserine Lactones as Quorum Sensing Modulators

    PubMed Central

    Stacy, Danielle M.; Le Quement, Sebastian T.; Hansen, Casper L.; Clausen, Janie W.; Tolker-Nielsen, Tim; Brummond, Jacob W.; Givskov, Michael; Nielsen, Thomas E.; Blackwell, Helen E.

    2013-01-01

    Many bacterial species are capable of assessing their local population densities through a cell-cell signaling mechanism termed quorum sensing (QS). This intercellular communication process is mediated by small molecule or peptide ligands and their cognate protein receptors. Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on a host. Consequently, approaches to intercept QS have attracted considerable attention as potential anti-infective therapies. Our interest in the development of small molecule tools to modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl L-homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and purities using efficient Cu(I)-catalyzed azide-alkyne couplings. These compounds were evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens – LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii – using bacterial reporter strains. Several triazole derivatives were identified that were capable of strongly modulating the activity of LasR and AbaR. These compounds represent a new and synthetically accessible class of AHL analogs, and could find utility as chemical tools to study QS and its role in bacterial virulence. PMID:23258305

  2. Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group.

    PubMed

    Schikora, Adam; Schenk, Sebastian T; Hartmann, Anton

    2016-04-01

    Bacterial quorum sensing (QS) mechanisms play a crucial role in the proper performance and ecological fitness of bacterial populations. Many key physiological processes are regulated in a QS-dependent manner by auto-inducers, like the N-acyl homoserine lactones (AHLs) in numerous Gram-negative bacteria. In addition, also the interaction between bacteria and eukaryotic hosts can be regulated by AHLs. Those mechanisms gained much attention, because of the positive effects of different AHL molecules on plants. This positive impact ranges from growth promotion to induced resistance and is quite contrasting to the rather negative effects observed in the interactions between bacterial AHL molecules and animals. Only very recently, we began to understand the molecular mechanisms underpinning plant responses to AHL molecules. In this review, we gathered the latest information in this research field. The first part gives an overview of the bacterial aspects of quorum sensing. Later we focus on the impact of AHLs on plant growth and AHL-priming, as one of the most understood phenomena in respect to the inter-kingdom interactions based on AHL-quorum sensing molecules. Finally, we discuss the potential benefits of the understanding of bacteria-plant interaction for the future agricultural applications.

  3. Diversity and N-acyl-homoserine lactone production by Gammaproteobacteria associated with Avicennia marina rhizosphere of South Indian mangroves.

    PubMed

    Viswanath, Ganga; Jegan, Sekar; Baskaran, Viswanathan; Kathiravan, Raju; Prabavathy, Vaiyapuri Ramalingam

    2015-07-01

    The diversity of N-acyl-homoserine lactone (AHL)-producing rhizosphere bacterial community associated with Avicennia marina in the mangrove ecosystems of South India was investigated. Approximately 800 rhizobacteria were isolated from A. marina, and they were screened for the production of AHL using two biosensors, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 (pZLR4). Among the total isolates screened, 7% of the rhizobacteria showed positive induction for AHL signals. The BOX-PCR profile of 56 positive isolates represented 11 distinct genotypic groups. Phylogenetic analyses of the 16S rRNA sequences of 16 representatives showed that the isolates belonged to the class Gammaproteobacteria, which represented six different genera: Pseudomonas, Aeromonas, Vibrio, Photobacterium, Serratia and Halomonas. The study also identified three AHL-producing species, namely, Photobacterium halotolerans MSSRF QS48, Vibrio xiamenensis MSSRF QS47 and Pseudomonas sp. MSSRF QS1 that had not been reported previously. AHL profiling by TLC detected short chains C4, C6 and C8-HSL, and long chains C10 and C12-HSL with both unsubstituted and substituted side chains among the 16 representative AHL positives. This is the first report concerning the diversity of AHL-producing Gammaproteobacteria from mangrove ecosystems exhibiting diverse AHL profiles.

  4. Sustained Pax6 Expression Generates Primate-like Basal Radial Glia in Developing Mouse Neocortex

    PubMed Central

    Mora-Bermúdez, Felipe; Taverna, Elena; Haffner, Christiane; Fu, Jun; Anastassiadis, Konstantinos; Stewart, A. Francis; Huttner, Wieland B.

    2015-01-01

    The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21–CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex. PMID:26252244

  5. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse.

    PubMed

    Rahimi Balaei, Maryam; Jiao, Xiaodan; Ashtari, Niloufar; Afsharinezhad, Pegah; Ghavami, Saeid; Marzban, Hassan

    2016-01-15

    Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax--naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration.

  6. Sustained Pax6 Expression Generates Primate-like Basal Radial Glia in Developing Mouse Neocortex.

    PubMed

    Wong, Fong Kuan; Fei, Ji-Feng; Mora-Bermúdez, Felipe; Taverna, Elena; Haffner, Christiane; Fu, Jun; Anastassiadis, Konstantinos; Stewart, A Francis; Huttner, Wieland B

    2015-08-01

    The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21-CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex.

  7. Differences in gene expression between mouse and human for dynamically regulated genes in early embryo.

    PubMed

    Madissoon, Elo; Töhönen, Virpi; Vesterlund, Liselotte; Katayama, Shintaro; Unneberg, Per; Inzunza, Jose; Hovatta, Outi; Kere, Juha

    2014-01-01

    Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.

  8. Synthesis, characterization and stereochemistry of N-acyl-r-2,c-4-bis(4-methoxyphenyl)-3-azabicyclo[3.3.1]nonanes

    NASA Astrophysics Data System (ADS)

    Akila, A.; Ponnuswamy, S.; Shreevidhyaa Suressh, V.; Usha, G.

    2015-08-01

    Four new N-acyl-2,4-bis(4-methoxyphenyl)-3-azabicyclo[3.3.1]nonanes 3-6 have been synthesized. The structural characterization and the conformational preferences of the compounds 3-6 have been carried out using IR, 1D and 2D NMR and Mass spectral data. The NMR spectral data indicates that the N-acyl-2,4-bis(4-methoxyphenyl)-3-azabicyclo[3.3.1]nonanes 3-6 prefer to exist in twin-chair conformation with partial flattening at nitrogen end. To avoid A1,3-strain, the anisyl groups at C2 and C4 are forced to occupy the quasi-axial orientation. X-ray crystal structure of the N-dichloroacetyl-2,4-bis(4-methoxyphenyl)-3-azabicyclo[3.3.1]nonane 6 also supports the similar conformation in the solid state.

  9. HBV life cycle is restricted in mouse hepatocytes expressing human NTCP

    PubMed Central

    Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

    2014-01-01

    Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1–6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1–6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created. PMID:24509445

  10. Glycogen phosphorylase as a target for type 2 diabetes: synthetic, biochemical, structural and computational evaluation of novel N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors.

    PubMed

    Kantsadi, Anastassia L; Parmenopoulou, Vanessa; Bakalov, Dimitar N; Snelgrove, Laura; Stravodimos, George A; Chatzileontiadou, Demetra S M; Manta, Stella; Panagiotopoulou, Angeliki; Hayes, Joseph M; Komiotis, Dimitri; Leonidas, Demetres D

    2015-01-01

    Glycogen phosphorylase (GP), a validated target for the development of anti-hyperglycaemic agents, has been targeted for the design of novel glycopyranosylamine inhibitors. Exploiting the two most potent inhibitors from our previous study of N-acyl-β-D-glucopyranosylamines (Parmenopoulou et al., Bioorg. Med. Chem. 2014, 22, 4810), we have extended the linking group to -NHCONHCO- between the glucose moiety and the aliphatic/aromatic substituent in the GP catalytic site β-cavity. The N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors were synthesized and their efficiency assessed by biochemical methods, revealing inhibition constant values of 4.95 µM and 2.53 µM. Crystal structures of GP in complex with these inhibitors were determined and analyzed, providing data for further structure based design efforts. A novel Linear Response - Molecular Mechanics Coulomb Surface Area (LR-MM-CBSA) method has been developed which relates predicted and experimental binding free energies for a training set of N-acyl-N´-(β-D-glucopyranosyl) urea ligands with a correlation coefficient R(2) of 0.89 and leave-one-out cross-validation (LOO-cv) Q(2) statistic of 0.79. The method has significant applications to direct future lead optimization studies, where ligand entropy loss on binding is revealed as a key factor to be considered. ADMET property predictions revealed that apart from potential permeability issues, the synthesized N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors have drug-like potential without any toxicity warnings.

  11. Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis.

    PubMed

    Wang, Fei; Hu, Jiarui; Song, Ping; Gong, Wuming

    2007-12-01

    The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

  12. Differential expression and regulation of angiopoietin-2 in mouse uterus during preimplantation period.

    PubMed

    Guo, Bin; Wang, Wei; Li, Shi-Jie; Han, Yu-Shuai; Zhang, Lu; Zhang, Xue-Ming; Liu, Ju-Xiong; Yue, Zhan-Peng

    2012-02-01

    Angiogenesis is crucial to successful implantation and decidualization, however, as an important angiogenic growth factor, the effect of Ang-2 in the process of implantation and decidualization is still unknown. This study is to investigate the differential expression of Ang-2 in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and RT-PCR. There is no detectable Ang-2 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On days 6-8, Ang-2 mRNA is mainly expressed in the primary decidua of mesometrial side, and the expression gradually increases. By RT-PCR, a significantly higher level of Ang-2 expression is observed on day 8 of pregnancy, although Ang-2 expression can be found through days 1-8. Similarly, Ang-2 is highly expressed in decidualized cells under artificial decidualization. In the ovariectomized mouse uterus, Ang-2 expression gradually increases after estrogen injection and with peak levels at 12 hr, while progesterone injection can cause a decline in uterine Ang-2 mRNA level, which reaches a nadir at 12 hr. These results suggest that Ang-2 may play a key role in the process of mouse decidualization. Estrogen can induce the expression of Ang-2 while progesterone can inhibit its expression in the ovariectomized mouse uterus.

  13. Digital Gene Expression Tag Profiling Analysis of the Gene Expression Patterns Regulating the Early Stage of Mouse Spermatogenesis

    PubMed Central

    Meng, Lijun; Liu, Meiling; Zhao, Lina; Hu, Fen; Ding, Cunbao; Wang, Yang; He, Baoling; Pan, Yuxin; Fang, Wei; Chen, Jing; Hu, Songnian; Jia, Mengchun

    2013-01-01

    Detailed characterization of the gene expression patterns in spermatogonia and primary spermatocytes is critical to understand the processes which occur prior to meiosis during normal spermatogenesis. The genome-wide expression profiles of mouse type B spermatogonia and primary spermatocytes were investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early spermatogenesis were systematically analyzed. Gene expression patterns vary significantly between mouse type B spermatogonia and primary spermatocytes. The functional analysis revealed that genes related to junction assembly, regulation of the actin cytoskeleton and pluripotency were most significantly differently expressed. Pathway analysis indicated that the Wnt non-canonical signaling pathway played a central role and interacted with the actin filament organization pathway during the development of spermatogonia. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of early spermatogenesis. PMID:23554914

  14. Oxytocin receptor is differentially expressed in mouse endometrium and embryo during blastocyst implantation.

    PubMed

    Beretsos, Panagiotis; Loutradis, Dimitris; Koussoulakos, Stauros; Margaritis, Loukas H; Kiapekou, Erasmia; Mastorakos, George; Papaspirou, Irini; Makris, Nikolaos; Makrigiannakis, Antonis; Antsaklis, Aris

    2006-12-01

    The oxytocin (OT)-oxytocin receptor (OTR) system of the mammalian uterus has mainly been studied in relation to its involvement in the onset of labor. The aim of this study was to elucidate the in vivo expression and localization pattern of OTR in the mouse endometrium and embryo during implantation, as well as OTR mRNA expression in the in vitro developing mouse embryo. The expression of OTR or OT was detected immunohistochemically in uterine tissue sections of 5- to 8-week-old female mice between days 4 and 10 of an established pregnancy. In addition, the expression of OTR mRNA was detected by means of reverse transcription polymerase chain reaction (RT-PCR) in mouse oocytes and embryos up to the blastocyst stage. The mean ratios of normalized expression levels of OTR gene in all samples were also calculated. The recorded increase in OTR mRNA immediately after fertilization could mean a possible role of OT in this process, as OTR mRNA gradually decreased after the four-cell stage of pre-embryonic development. The differential expression of OTR during embryonic apposition and embryonic invasion/placentation in the mouse uterus suggests a potential role of OT in the implantation process of the mouse. It is possible that the interaction of OTR with the hormones included in the ovulation induction regiments utilized today in in vitro fertilization (IVF) could be affecting the receptivity/quality of the implanting endometrium.

  15. Expression and regulation of Runx3 in mouse uterus during the peri-implantation period.

    PubMed

    Bai, Zhi-Kun; Guo, Bin; Tian, Xue-Chao; Li, Dang-Dang; Wang, Shou-Tang; Cao, Hang; Wang, Qu-Yuan; Yue, Zhan-Peng

    2013-10-01

    The aim of this study was to investigate the differential expression and regulation of Runt-related transcription factor 3 (Runx3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization. There was a low level of the Runx3 mRNA expression in the mouse uterus on days 1-4 of pregnancy. On day 5 when embryo implanted, Runx3 mRNA signal was obviously observed in the stromal cells surrounding the implanting blastocyst. From day 6 to 8 of pregnancy, Runx3 mRNA was highly expressed in the decidual cells and mesometrial decidual beds. Similarly, Runx3 mRNA was strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Runx3 mRNA signal was detected in the uterus with activated implantation. In the ovariectomized mouse uterus, estrogen could induce the expression of Runx3, while progesterone had no effects. These results suggest that Runx3 may play an important role during mouse implantation and decidualization. Estrogen can induce the expression of Runx3 in the ovariectomized mouse uterus.

  16. A novel plasmid for detection of N-acyl homoserine lactones.

    PubMed

    Ling, Elizabeth A; Ellison, Matthew L; Pesci, Everett C

    2009-07-01

    Many bacteria utilize acyl-homoserine lactones as cell to cell signals that can regulate the expression of numerous genes. Structural differences in acyl-homoserine lactones produced by different bacteria, such as acyl side chain length and the presence or absence of an oxy group, make many of the commonly used detection bioassays impractical for broad range detection. Here we present a simple, broad range acyl-homoserine lactone detection bioassay that can be used to detect a wide range of these chemical signals. A plasmid (pEAL01) was constructed and transformed into Pseudomonas aeruginosa strain QSC105 to allow for detection of a broad range of acyl-homoserine lactones through induction of a lasB'-lacZ transcriptional fusion. Monitoring beta-galactosidase activity from this bioassay showed that P. aeruginosa strain QSC105 (pEAL01) could detect the presence of eight acyl-homoserine lactones tested at physiological concentrations. This novel strain could also detect acyl-homoserine lactones from the extracts of four different bacteria that produce different acyl-homoserine lactones signals. These data indicate that strain QSC105 (pEAL01) can be used to detect a wide variety of acyl-homoserine lactones by a simple beta-galactosidase assay and this bioassay could be a useful and inexpensive tool to quickly identify the presence of these signal molecules.

  17. Cocaine-induced behavioral sensitization decreases the expression of endocannabinoid signaling-related proteins in the mouse hippocampus.

    PubMed

    Blanco, Eduardo; Galeano, Pablo; Palomino, Ana; Pavón, Francisco J; Rivera, Patricia; Serrano, Antonia; Alen, Francisco; Rubio, Leticia; Vargas, Antonio; Castilla-Ortega, Estela; Decara, Juan; Bilbao, Ainhoa; de Fonseca, Fernando Rodríguez; Suárez, Juan

    2016-03-01

    In the reward mesocorticolimbic circuits, the glutamatergic and endocannabinoid systems are implicated in neurobiological mechanisms underlying cocaine addiction. However, the involvement of both systems in the hippocampus, a critical region to process relational information relevant for encoding drug-associated memories, in cocaine-related behaviors remains unknown. In the present work, we studied whether the hippocampal gene/protein expression of relevant glutamate signaling components, including glutamate-synthesizing enzymes and metabotropic and ionotropic receptors, and the hippocampal gene/protein expression of cannabinoid type 1 (CB1) receptor and endocannabinoid metabolic enzymes were altered following acute and/or repeated cocaine administration resulting in conditioned locomotion and locomotor sensitization. Results showed that acute cocaine administration induced an overall down-regulation of glutamate-related gene expression and, specifically, a low phosphorylation level of GluA1. In contrast, locomotor sensitization to cocaine produced an up-regulation of several glutamate receptor-related genes and, specifically, an increased protein expression of the GluN1 receptor subunit. Regarding the endocannabinoid system, acute and repeated cocaine administration were associated with an increased gene/protein expression of CB1 receptors and a decreased gene/protein expression of the endocannabinoid-synthesis enzymes N-acyl phosphatidylethanolamine D (NAPE-PLD) and diacylglycerol lipase alpha (DAGLα). These changes resulted in an overall decrease in endocannabinoid synthesis/degradation ratios, especially NAPE-PLD/fatty acid amide hydrolase and DAGLα/monoacylglycerol lipase, suggesting a reduced endocannabinoid production associated with a compensatory up-regulation of CB1 receptor. Overall, these findings suggest that repeated cocaine administration resulting in locomotor sensitization induces a down-regulation of the endocannabinoid signaling that could

  18. Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues.

    PubMed Central

    Hooper, John D; Campagnolo, Luisa; Goodarzi, Goodarz; Truong, Tony N; Stuhlmann, Heidi; Quigley, James P

    2003-01-01

    We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial

  19. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  20. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

    PubMed

    Nemoto, Takayuki K; Ohara-Nemoto, Yuko; Bezerra, Gustavo Arruda; Shimoyama, Yu; Kimura, Shigenobu

    2016-03-11

    Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.

  1. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides*

    PubMed Central

    Nemoto, Takayuki K.; Ohara-Nemoto, Yuko; Bezerra, Gustavo Arruda; Shimoyama, Yu; Kimura, Shigenobu

    2016-01-01

    Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser615 and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm−1 s−1, optimal pH was 7–8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met16–Glu101). Three-dimensional modeling revealed the three domain structures (residues Met16–Ala126, which has no similar homologue with known structure; residues Leu127–Met495 (β-propeller domain); and residues Ala496–Phe736 (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides. PMID:26733202

  2. Quorum sensing of bacteria and trans-kingdom interactions of N-acyl homoserine lactones with eukaryotes.

    PubMed

    Hartmann, Anton; Schikora, Adam

    2012-06-01

    Many environmental and interactive important traits of bacteria, such as antibiotic, siderophore or exoenzyme (like cellulose, pectinase) production, virulence factors of pathogens, as well as symbiotic interactions, are regulated in a population density-dependent manner by using small signaling molecules. This phenomenon, called quorum sensing (QS), is widespread among bacteria. Many different bacterial species are communicating or "speaking" through diffusible small molecules. The production often is sophisticatedly regulated via an autoinducing mechanism. A good example is the production of N-acyl homoserine lactones (AHL), which occur in many variations of molecular structure in a wide variety of Gram-negative bacteria. In Gram-positive bacteria, other compounds, such as peptides, regulate cellular activity and behavior by sensing the cell density. The degradation of the signaling molecule--called quorum quenching--is probably another important integral part in the complex quorum sensing circuit. Most interestingly, bacterial quorum sensing molecules also are recognized by eukaryotes that are colonized by QS-active bacteria. In this case, the cross-kingdom interaction can lead to specific adjustment and physiological adaptations in the colonized eukaryote. The responses are manifold, such as modifications of the defense system, modulation of the immune response, or changes in the hormonal status and growth responses. Thus, the interaction with the quorum sensing signaling molecules of bacteria can profoundly change the physiology of higher organisms too. Higher organisms are obligatorily associated with microbial communities, and these truly multi-organismic consortia, which are also called holobionts, can actually be steered via multiple interlinked signaling substances that originate not only from the host but also from the associated bacteria.

  3. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean.

    PubMed

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants' pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  4. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean

    PubMed Central

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants’ pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  5. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  6. Effects of CHO-expressed recombinant lactoferrins on mouse dendritic cell presentation and function.

    PubMed

    Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

    2015-07-01

    Lactoferrin (LF), a natural iron-binding protein, has previously demonstrated effectiveness in enhancing the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine. This report investigates immune modulatory effects of Chinese hamster ovary (CHO) cell-expressed recombinant mouse and human LFs on mouse bone marrow-derived dendritic cells (BMDCs), comparing homologous and heterologous functions. BCG-infected BMDCs were cultured with LF, and examined for class II presentation molecule expression. Culturing of BCG-infected BMDCs with either LF decreased the class II molecule-expressing population. Mouse LF significantly increased the production of IL-12p40, IL-1β and IL-10, while human LF-treated BMDCs increased only IL-1β and IL-10. Overlaying naïve CD4 T-cells onto BCG-infected BMDCs cultured with mouse LF increased IFN-γ, whereas the human LF-exposed group increased IFN-γ and IL-17 from CD4 T cells. Overlay of naïve CD8 T cells onto BCG-infected BMDCs treated with mouse LF increased the production of IFN-γ and IL-17, while similar experiments using human LF only increased IL-17. This report is the first to examine mouse and human recombinant LFs in parallel experiments to assess murine DC function. These results detail the efficacy of the human LF counterpart used in a heterologous system to understand LF-mediated events that confer BCG efficacy against Mycobacterium tuberculosis challenge.

  7. Differential expression and regulation of angiopoietin-3 in mouse uterus during preimplantation period.

    PubMed

    Guo, Bin; Zhang, Xue-Ming; Li, Shi-Jie; Tian, Xue-Chao; Wang, Shou-Tang; Li, Dang-Dang; Liu, Ju-Xiong; Yue, Zhan-Peng

    2012-06-01

    Angiogenesis is necessary for successful implantation and decidualization. This study was to investigate the differential expression of angiopoietin-3 (Ang-3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). There was no detectable Ang-3 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On day 6 of pregnancy, a low level of Ang-3 mRNA signal was seen in the primary decidua. Ang-3 mRNA expression gradually increased on days 7 and 8 of pregnancy along with the development of decidua, and its expression scope was also expanded. The RT-PCR result indicated that Ang-3 mRNA expression was low on days 1-4 of pregnancy. On day 5, as embryo implanted, Ang-3 mRNA was highly expressed in mouse uterus, and the expression gradually increased on days 6-8 of pregnancy, with peak level on day 8 of pregnancy. Similarly, Ang-3 mRNA was also strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Ang-3 mRNA expression was detected in activated implantation uterus by RT-PCR. In the ovariectomized mouse uterus, Ang-3 mRNA expression increased and reached the highest level at 12 hr after injection of estrogen, progesterone, and estrogen plus progesterone, respectively. These results suggest that Ang-3 may play an important role during the process of mouse decidualization. Both estrogen and progesterone can induce the expression of Ang-3 in ovariectomized mouse uterus.

  8. Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ

    PubMed Central

    Ackels, Tobias; von der Weid, Benoît; Rodriguez, Ivan; Spehr, Marc

    2014-01-01

    The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology. PMID:25484858

  9. Differential expression and regulation of Cryab in mouse uterus during preimplantation period.

    PubMed

    Tian, Xue-Chao; Wang, Qu-Yuan; Li, Dang-Dang; Wang, Shou-Tang; Yang, Zhan-Qing; Guo, Bin; Yue, Zhan-Peng

    2013-06-01

    The aim of this study was to examine the expression and regulation of the crystallin, alpha B (Cryab) gene in mouse uterus during the peri-implantation period by in situ hybridization and real-time PCR. There was no detectable Cryab mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy when embryo implanted, a high level of Cryab mRNA signal was found in the subluminal stroma surrounding the implanting blastocyst. On days 6-8, Cryab mRNA was strongly expressed in the primary decidua. By real-time PCR, a high level of Cryab expression was detected on days 7 and 8 of pregnancy, although Cryab expression was seen from days 1 to 8. Under in vivo and in vitro artificial decidualization, Cryab expression was significantly elevated. Compared with the progesterone-primed delayed implantation uterus, a high level of Cryab mRNA expression was observed in estrogen-activated implantation uterus. In the uterine stromal cells, cAMP, estrogen, and progesterone could induce the expression of Cryab gene. In the ovariectomized mouse uterus, estrogen could also induce the expression of Cryab while progesterone inhibited its expression. Our data suggest that Cryab may play an important role during mouse embryo implantation and decidualization and that estrogen and progesterone can regulate the expression of Cryab gene.

  10. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines

    PubMed Central

    Yamamizu, Kohei; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B.; Schlessinger, David; Ko, Minoru S. H.

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range – and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this “NIA Mouse ESC Bank,” we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  11. Gene activation properties of a mouse DNA sequence isolated by expression selection.

    PubMed Central

    von Hoyningen-Huene, V; Norbury, C; Griffiths, M; Fried, M

    1986-01-01

    The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences. Mes-1 has been tested in long-term transformation and short-term CAT expression assays. In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene. The element is active with heterologous promoters and functions efficiently in both rat and mouse cells. MES-1 activates expression by increasing transcription from the test gene's own start (cap) site. Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer-like properties from total cellular DNA. Images PMID:3016657

  12. Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

    PubMed Central

    Bono, Hidemasa; Yagi, Ken; Kasukawa, Takeya; Nikaido, Itoshi; Tominaga, Naoko; Miki, Rika; Mizuno, Yosuke; Tomaru, Yasuhiro; Goto, Hitoshi; Nitanda, Hiroyuki; Shimizu, Daisuke; Makino, Hirochika; Morita, Tomoyuki; Fujiyama, Junshin; Sakai, Takehito; Shimoji, Takashi; Hume, David A.; Hayashizaki, Yoshihide; Okazaki, Yasushi

    2003-01-01

    The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed. PMID:12819129

  13. Basigin expression and hormonal regulation in mouse uterus during the peri-implantation period.

    PubMed

    Xiao, Li-Juan; Chang, Hong; Ding, Nai-Zheng; Ni, Hua; Kadomatsu, Kenji; Yang, Zeng-Ming

    2002-09-01

    Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has been shown to be essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of basigin gene in mouse uterus during the peri-implantation period. Basigin immunostaining and mRNA were strongly localized in luminal and glandular epithelium on day 1 of pregnancy and gradually decreased to a basal level from day 2-4 of pregnancy. Basigin mRNA expression in the sub-luminal stroma was first detected on day 3 of pregnancy and increased on day 4 of pregnancy. On day 5 of pregnancy, the expression of basigin protein and mRNA was only detected in the implanting embryos, and the luminal epithelium and sub-luminal stroma surrounding the embryos. A similar expression pattern of basigin was also induced in the delayed-implantation uterus which was activated by estrogen injection. On day 6-8 of pregnancy, although a basal level of basigin protein was detected in the secondary decidual zone, basigin mRNA expression was strongly seen in this location. Basigin mRNA was also highly expressed in the decidualized cells under artificial decidualization. Estrogen significantly stimulated basigin expression in the ovariectomized mouse uterus. A high level of basigin immunostaining and mRNA was also seen in proestrus and estrus uteri. These results suggest that basigin expression is closely related to mouse implantation and up-regulated by estrogen.

  14. Connexin expression in epidermal cell lines from SENCAR mouse skin tumors.

    PubMed

    Budunova, I V; Carbajal, S; Viaje, A; Slaga, T J

    1996-03-01

    Alteration of gap-junctional intercellular communication (GJIC) has long been proposed to be involved in carcinogenesis. Previously, we reported that the level of gap junctional intercellular communication in mouse skin carcinoma cell lines is significantly lower than in papilloma cell lines and normal mouse keratinocytes Klann et al., Cancer Res 49:699-705, 1989). Here, we present data on expression of the gap-junctional protein connexins (Cx) 26, Cx31.1, and Cx43 in a comprehensive panel of keratinocyte cell lines representing different stages of mouse skin carcinogenesis and the effect of different conditions of propagation on Cx phenotype. Northern and western blot analyses and immunostaining showed that all cell lines studied in vitro expressed Cx43 but most did not express Cx31.1 or Cx26. The abundance of Cx43 expression on plasma membranes correlated well with the level of GJIC. In vivo expression of Cx43 and Cx26 was strongly increased. Whereas none of tumorigenic cell lines expressed Cx26 gap junctions in culture, those growing as tumors in nude mice began to express Cx26 protein. The comparison of Cx expression on the keratinocyte membranes in three different groups of tumors (papillomas and squamous cell and spindle cell carcinomas) clearly revealed that the abundance of Cx43 and Cx26 expression directly correlated with the level of tumor differentiation. All studied tumors were Cx31.1 negative. These results suggest that both Cx expression and gap-junction permeability are gradually reduced during the tumor progression stage of mouse skin carcinogenesis.

  15. A bioluminescence reporter mouse that monitors expression of constitutively active β-catenin

    PubMed Central

    Kommagani, Ramakrishna; Peavey, Mary C.; Hai, Lan; Lonard, David M.; Lydon, John P.

    2017-01-01

    This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse—when crossed with the appropriate effector transgenic—will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues. PMID:28253313

  16. A gene expression resource generated by genome-wide lacZ profiling in the mouse

    PubMed Central

    Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A.; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L.; Wardle-Jones, Hannah; Carragher, Damian M.; Karp, Natasha A.; Smedley, Damian; Adams, Niels C.; Bussell, James N.; Adams, David J.; Ramírez-Solis, Ramiro; Steel, Karen P.; Galli, Antonella; White, Jacqueline K.

    2015-01-01

    ABSTRACT Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

  17. Assessment of a systematic expression profiling approach in ENU-induced mouse mutant lines.

    PubMed

    Seltmann, Matthias; Horsch, Marion; Drobyshev, Alexei; Chen, Yali; de Angelis, Martin Hrabé; Beckers, Johannes

    2005-01-01

    Comparative genomewide expression profiling is a powerful tool in the effort to annotate the mouse genome with biological function. The systematic analysis of RNA expression data of mouse lines from the Munich ENU mutagenesis screen might support the understanding of the molecular biology of such mutants and provide new insights into mammalian gene function. In a direct comparison of DNA microarray experiments of individual versus pooled RNA samples of organs from ENU-induced mouse mutants, we provide evidence that individual RNA samples may outperform pools in some aspects. Genes with high biological variability in their expression levels (noisy genes) are identified as false positives in pooled samples. Evidence suggests that highly stringent housing conditions and standardized procedures for the isolation of organs significantly reduce biological variability in gene expression profiling experiments. Data on wild-type individuals demonstrate the positive effect of controlling variables such as social status, food intake before organ sampling, and stress with regard to reproducibility of gene expression patterns. Analyses of several organs from various ENU-induced mutant lines in general show low numbers of differentially expressed genes. We demonstrate the feasibility to detect transcriptionally affected organs employing RNA expression profiling as a tool for molecular phenotyping.

  18. Increased Expression of Hepatocyte Nuclear Factor 6 Stimulates Hepatocyte Proliferation during Mouse Liver Regeneration

    PubMed Central

    Tan, Yongjun; Yoshida, Yuichi; Hughes, Douglas E.; Costa, Robert H.

    2005-01-01

    Background & Aims The Hepatocyte Nuclear Factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for Chromatin Immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration. Methods Tail vein injection of adenovirus expressing the HNF6 cDNA (AdHNF6) was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by Bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters. Results Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S-phase) and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by siRNA transfection exhibit a 50% reduction in S-phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen Tumor Growth Factor α (TGFα) and the cell cycle regulators Cyclin D1 and Forkhead Box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that TGFα, Cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Co-immunoprecipitation assays with regenerating mouse liver extracts reveal association between HNF6 and Foxm1 proteins and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity. Conclusion These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes. PMID:16618419

  19. Temporal and spatial mouse brain expression of cereblon, an ionic channel regulator involved in human intelligence.

    PubMed

    Higgins, Joseph J; Tal, Adit L; Sun, Xiaowei; Hauck, Stefanie C R; Hao, Jin; Kosofosky, Barry E; Rajadhyaksha, Anjali M

    2010-03-01

    A mild form of autosomal recessive, nonsyndromal intellectual disability (ARNSID) in humans is caused by a homozygous nonsense mutation in the cereblon gene (mutCRBN). Rodent crbn protein binds to the intracellular C-terminus of the large conductance Ca(2+)-activated K(+)channel (BK(Ca)). An mRNA variant (human SITE 2 INSERT or mouse strex) of the BK(Ca) gene (KCNMA1) that is normally expressed during embryonic development is aberrantly expressed in mutCRBN human lymphoblastoid cell lines (LCLs) as compared to wild-type (wt) LCLs. The present study analyzes the temporal and spatial distribution of crbn and kcnma1 mRNAs in the mouse brain by the quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR). The spatial expression pattern of endogenous and exogenous crbn proteins is characterized by immunostaining. The results show that neocortical (CTX) crbn and kcnma1 mRNA expression increases from embryonic stages to adulthood. The strex mRNA variant is >3.5-fold higher in embryos and decreases rapidly postnatally. Mouse crbn mRNA is abundant in the cerebellum (CRBM), with less expression in the CTX, hippocampus (HC), and striatum (Str) in adult mice. The intracytoplasmic distribution of endogenous crbn protein in the mouse CRBM, CTX, HC, and Str is similar to the immunostaining pattern described previously for the BK(Ca) channel. Exogenous hemagglutinin (HA) epitope-tagged human wt- and mutCRBN proteins using cDNA transfection in HEK293T cell lines showed the same intracellular expression distribution as endogenous mouse crbn protein. The results suggest that mutCRBN may cause ARNSID by disrupting the developmental regulation of BK(Ca) in brain regions that are critical for memory and learning.

  20. Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear.

    PubMed

    Oesterle, Elizabeth C; Campbell, Sean; Taylor, Ruth R; Forge, Andrew; Hume, Clifford R

    2008-03-01

    Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

  1. Patterned expression of ion channel genes in mouse dorsal raphe nucleus determined with the Allen Mouse Brain Atlas

    PubMed Central

    Templin, J. Scott; Bang, Sun Jung; Soiza-Reilly, Mariano; Berde, Charles B.; Commons, Kathryn G.

    2012-01-01

    The dorsal raphe nucleus (DR) is the major source of serotonin (5-hydroxytryptamine, 5-HT) in the forebrain and dysfunction of this midbrain structure is implicated in affective disorders. The DR is composed of several types of 5-HT and non-5-HT neurons and their excitable-membrane properties are heterogeneous and overlapping. In order to understand how these properties may be generated, we examined the mRNA expression patterns of voltage- and ligand-gated ion channels in the DR using the Allen Mouse Brain Atlas. Since DR cytoarchitecture is organized with respect to the midline, we sought to identify genes that were expressed in a pattern with respect to the midline, either enriched or depleted, rather than those that were homogenously expressed throughout the DR. Less than 10% of the screened genes for voltage-gated ion channels showed patterned expression within the DR. Identified genes included voltage-gated sodium channel beta subunits, potassium channels, P/Q-, N-type calcium channels, as well as the alpha2/delta-1 calcium channel. Several voltage-gated chloride channels were also identified, although these may function within intracellular compartments. Of the ligand-gated ion channels examined, 20% showed patterned expression. These consisted primarily of glutamate and GABA-A receptor subunits. The identified genes likely contribute to unique excitable properties of different groups of neurons in the DR and may include novel pharmacologic targets for affective disorders. PMID:22534482

  2. Expression of gamma-glutamyl transpeptidase in midgestation mouse yolk sac and mouse visceral yolk sac carcinoma cells.

    PubMed

    Sepulveda, A R; Habib, G M; Damjanov, A; Matacic, S; Damjanov, I; Lebovitz, R M; Lieberman, M W

    1995-08-01

    gamma-Glutamyl transpeptidase (gamma GT) is a crucial enzyme for the metabolism of xenobiotics and endogenous mediators of biological functions (leukotrienes, prostaglandins, and hepoxillins). Yet little is known about its potential role during development. It is a single copy gene expressed from at least seven promoters. Using histochemistry and immunohistochemistry we demonstrate that gamma GT first appears in the midgestational yolk sacs of mouse embryos. Established cell lines with phenotypic features of yolk sac endoderm (JC-44) or embryonic stem cells were also assayed for the expression of gamma GT. Significant levels were detected in JC-44 cells and higher levels were found in JC-44-derived embryoid bodies. Because this cell line appears to be a good in vitro counterpart of yolk sac differentiation, we characterized the gamma GT mRNA types expressed in JC-44 cells. By ribonuclease protection analysis, gamma GT RNA types IV and VI represent about 80% of the total gamma GT RNA in JC-44 embryoid bodies. Reverse transcription-mediated polymerase chain reaction detected low amounts of gamma GT RNA types I, III, and V. Expression of gamma GT in yolk sac follows a pattern seen in many tissues in which one or two gamma GT RNA types dominate the expression profile; however, the reason for this tissue specificity is unknown.

  3. Heart valve cardiomyocytes of mouse embryos express the serotonin transporter SERT

    SciTech Connect

    Pavone, Luigi Michele Spina, Anna; Lo Muto, Roberta; Santoro, Dionea; Mastellone, Vincenzo; Avallone, Luigi

    2008-12-12

    Multiple evidence demonstrate a role for serotonin and its transporter SERT in heart valve development and disease. By utilizing a Cre/loxP system driven by SERT gene expression, we recently demonstrated a regionally restricted distribution of SERT-expressing cells in developing mouse heart. In order to characterize the cell types exhibiting SERT expression within the mouse heart valves at early developmental stages, in this study we performed immunohistochemistry for Islet1 (Isl1) and connexin-43 (Cx-43) on heart sections from SERT{sup Cre/+};ROSA26R embryos previously stained with X-gal. We observed the co-localization of LacZ staining with Isl1 labelling in the outflow tract, the right ventricle and the conal region of E11.5 mouse heart. Cx-43 labelled cells co-localized with LacZ stained cells in the forming atrioventricular valves. These results demonstrate the cardiomyocyte phenotype of SERT-expressing cells in heart valves of the developing mouse heart, thus suggesting an active role of SERT in early heart valve development.

  4. Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

    PubMed

    Zhang, Li-Ying; Hua, Yu-Ping; An, Tie-Zhu; Zuo, Ru-Juan; Nie, Wei-Tian; Wang, Lian-Bang; Shan, Chun-Hua

    2012-01-01

    Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.

  5. Slit and robo expression in the developing mouse lung.

    PubMed

    Greenberg, James M; Thompson, Felisa Y; Brooks, Sherry K; Shannon, John M; Akeson, Ann L

    2004-06-01

    Mammalian lung development is mediated through complex interactions between foregut endoderm and surrounding mesenchyme. As airway branching progresses, the mesenchyme undergoes dramatic remodeling and differentiation. Little is understood about the mechanisms that direct mesenchymal organization during lung development. A screen for candidate genes mediating this process identified Slit, a ligand for the Roundabout (Robo) receptor previously associated with guidance of axonal projections during central nervous system development. Here, we demonstrate by in situ hybridization that two Slit genes (Slit-2 and Slit-3) and two Robo genes (Robo-1 and Robo-2) are expressed in fetal lung mesenchyme. Slit-2 and Robo-1 expression is present throughout mesenchyme at midgestation and is not detectable by newborn day 1. Slit-3 and Robo-2 expression is restricted to specific, complementary subsets of mesenchyme. Robo-2 is expressed in mesenchymal cells immediately adjacent to large airways, whereas Slit-3 expression predominates in mesenchyme remote from airway epithelium. The temporal and spatial distribution of Slit and Robo mRNAs indicate that these genes may direct the functional organization and differentiation of fetal lung mesenchyme.

  6. Sensitive Whole-Cell Biosensor Suitable for Detecting a Variety of N-Acyl Homoserine Lactones in Intact Rhizosphere Microbial Communities▿

    PubMed Central

    DeAngelis, Kristen M.; Firestone, Mary K.; Lindow, Steven E.

    2007-01-01

    To investigate quorum sensing in rhizosphere soil, a whole-cell biosensor, Agrobacterium tumefaciens(pAHL-Ice), was constructed. The biosensor responded to all N-acyl homoserine lactones (AHLs) tested, except C4 homoserine lactone, with a minimum detection limit of 10−12 M, as well as to both exogenously added AHLs and AHL-producing bacterial strains in soil. This highly sensitive biosensor reveals for the first time the increased AHL availability in intact rhizosphere microbial communities compared to that in bulk soil. PMID:17400771

  7. Thyroid receptor ligands. Part 8: Thyromimetics derived from N-acylated-alpha-amino acid derivatives displaying modulated pharmacological selectivity compared with KB-141.

    PubMed

    Garg, Neeraj; Li, Yi-Lin; Garcia Collazo, Ana Maria; Litten, Chris; Ryono, Denis E; Zhang, Minsheng; Caringal, Yolanda; Brigance, Robert P; Meng, Wei; Washburn, William N; Agback, Peter; Mellström, Karin; Rehnmark, Stefan; Rahimi-Ghadim, Mahmoud; Norin, Thomas; Grynfarb, Marlena; Sandberg, Johnny; Grover, Gary; Malm, Johan

    2007-08-01

    Based on the scaffold of the pharmacologically selective thyromimetic 2b, structurally a close analog to KB-141 (2a), a number of novel N-acylated-alpha-amino acid derivatives were synthesized and tested in a TR radioligand binding assay as well as in a reporter cell assay. On the basis of TRbeta(1)-isoform selectivity and affinity, as well as affinity to the reporter cell assay, 3d was selected for further studies in the cholesterol-fed rat model. In this model 3d revealed an improved therapeutic window between cholesterol and TSH lowering but decreased margins versus tachycardia compared with 2a.

  8. Insights into the Genome Sequences of an N-Acyl Homoserine Lactone Molecule Producing Two Pseudomonas spp. Isolated from the Arctic

    PubMed Central

    Dharmaprakash, Akhilandeswarre; Reghunathan, Dinesh; Sivakumar, Krishnakutty C.; Prasannakumar, Manoj

    2016-01-01

    We report for the first time the draft genome sequence of two psychrotrophic Pseudomonas species, Pseudomonas simiae RGCB 73 and Pseudomonas brenneri RGCB 108, from the Arctic that produce more than one acyl homoserine lactone molecule of varied N-acyl length. The study confirms the presence of a LuxR-LuxI (type) mediated quorum-sensing system in both the Pseudomonas species and enables us to understand the role of quorum sensing in their survival in extremely cold environments. PMID:27491995

  9. Developmental expression of Sp1 in the mouse.

    PubMed Central

    Saffer, J D; Jackson, S P; Annarella, M B

    1991-01-01

    The expression of the trans-acting transcription factor Sp1 in mice was defined by a combination of RNA analysis and immunohistochemical localization of the Sp1 protein. Although ubiquitously expressed, there was an unexpected difference of at least 100-fold in the amount of Sp1 message in different cell types. Sp1 protein levels showed corresponding marked differences. Substantial variations in Sp1 expression were also found in some cell types at different stages of development. Sp1 levels appeared to be highest in developing hematopoietic cells, fetal cells, and spermatids, suggesting that an elevated Sp1 level is associated with the differentiation process. These results indicate that Sp1 has a regulatory function in addition to its general role in the transcription of housekeeping genes. Images PMID:2005904

  10. EMAGE: a spatial database of gene expression patterns during mouse embryo development

    PubMed Central

    Christiansen, Jeffrey H.; Yang, Yiya; Venkataraman, Shanmugasundaram; Richardson, Lorna; Stevenson, Peter; Burton, Nicholas; Baldock, Richard A.; Davidson, Duncan R.

    2006-01-01

    EMAGE () is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods. PMID:16381949

  11. EMAGE: a spatial database of gene expression patterns during mouse embryo development.

    PubMed

    Christiansen, Jeffrey H; Yang, Yiya; Venkataraman, Shanmugasundaram; Richardson, Lorna; Stevenson, Peter; Burton, Nicholas; Baldock, Richard A; Davidson, Duncan R

    2006-01-01

    EMAGE (http://genex.hgu.mrc.ac.uk/Emage/database) is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods.

  12. Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development.

    PubMed

    Chen, Li; Nakai, Masaaki; Belton, Robert J; Nowak, Romana A

    2007-02-01

    Mouse embryo implantation is a highly invasive and controlled process that involves remodeling and degradation of the extracellular matrix of the uterus. Matrix metalloproteinases (MMPs) are the main proteinases facilitating this process. Extracellular matrix metalloproteinase inducer (EMMPRIN) can stimulate the production of MMPs and is required for successful implantation in the mouse. The aims of the present study were to examine the expression profiles of mRNA and proteins for EMMPRIN and MMPs in the developing mouse embryo in vitro, and to study whether EMMPRIN protein induces the production of MMPs by mouse blastocysts. EMMPRIN mRNA, detected by RT-PCR, was present at all stages of embryo development from the one-cell to the blastocyst outgrowth. EMMPRIN protein, observed by confocal microscopy, was present on the cell surface at the same stages of development as was the mRNA. Of seven MMPs studied, murine collagenase-like A (Mcol-A), murine collagenase-like B (Mcol-B) and gelatinase A (MMP-2) mRNAs were detected only in blastocyst outgrowths by RT-PCR. Gelatinase B (MMP-9) mRNA was detected both in expanded blastocysts and blastocyst outgrowths. MMP-2 and -9 proteins were detected in the cytoplasm of outgrowing trophoblast cells. Collagenase-2 (MMP-8), collagenase-3 (MMP-13), or stromelysin-1 (MMP-3) mRNAs were not present at any stage of pre- or peri-implantation mouse embryo development. Quantitative RT-PCR analyses showed that recombinant EMMPRIN protein did not stimulate MMP-2 or -9 expression by mouse blastocyst outgrowths. These data suggest that EMMPRIN may regulate physiological functions other than MMP production by mouse embryos during implantation.

  13. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    SciTech Connect

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing . E-mail: jnzhang@dlmedu.edu.cn

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  14. Dynamic expression and heterogeneous intracellular location of En-1 during late mouse embryonic development.

    PubMed

    Zhong, Shan-chuan; Chen, Xing-shu; Cai, Qi-yan; Luo, Xue; Chen, Xing-hua; Liu, Jing; Yao, Zhong-xiang

    2010-01-01

    Engrailed-1 (En-1) is a transcription factor involved in the development of the midbrain/hindbrain during mouse early embryogenesis. Although En-1 is expressed from embryogenesis to adulthood, there has been no detailed description of its expression during late mouse embryonic development. Here we report the expression pattern of En-1 in the mouse embryo from E10.5 to the neonatal state. With immunohistochemistry we found that En-1 was expressed in the central nervous system (CNS) from E10.5 to the neonatal state, mostly restricted to the midbrain/hindbrain junction. Outside the CNS, En-1 is dynamically expressed in several neural crest-associated structures including the cranial mesenchyme, the mandibular arches, the vagus nerve, the dorsal root ganglia, the sympathetic ganglia, the somites, the heart and the cloaca. Additionally, we found that in the CNS, most of the En-1 was located in the nuclei, while outside the CNS, En-1 was mainly expressed in the cytoplasm. These findings provided additional evidence that En-1 may be involved in the development of neural crest cells.

  15. Transcriptional Profiling of mRNA Expression in the Mouse Distal Colon

    PubMed Central

    HOOGERWERF, WILLEMIJNTJE A.; SINHA, MALA; CONESA, ANA; LUXON, BRUCE A.; SHAHINIAN, VAHAKN B.; CORNÉLISSEN, GERMAINE; HALBERG, FRANZ; BOSTWICK, JONATHON; TIMM, JOHN; CASSONE, VINCENT M.

    2009-01-01

    Background & Aims Intestinal epithelial cells and the myenteric plexus of the mouse gastrointestinal tract contain a circadian clock–based intrinsic timekeeping system. Because disruption of the biological clock has been associated with increased susceptibility to colon cancer and gastrointestinal symptoms, we aimed to identify rhythmically expressed genes in the mouse distal colon. Methods Microarray analysis was used to identify genes that were rhythmically expressed over a 24-hour light/dark cycle. The transcripts were then classified according to expression pattern, function, and association with physiologic and pathophysiologic processes of the colon. Results A circadian gene expression pattern was detected in approximately 3.7% of distal colonic genes. A large percentage of these genes were involved in cell signaling, differentiation, and proliferation and cell death. Of all the rhythmically expressed genes in the mouse colon, approximately 7% (64/906) have been associated with colorectal cancer formation (eg, B-cell leukemia/lymphoma-2 [Bcl2]) and 1.8% (18/906) with various colonic functions such as motility and secretion (eg, vasoactive intestinal polypeptide, cystic fibrosis transmembrane conductance regulator). Conclusions A subset of genes in the murine colon follows a rhythmic expression pattern. These findings may have significant implications for colonic physiology and pathophysiology. PMID:18848557

  16. The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression.

    PubMed

    Mueller, Jacob L; Mahadevaiah, Shantha K; Park, Peter J; Warburton, Peter E; Page, David C; Turner, James M A

    2008-06-01

    According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)--the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells.

  17. Differential expression and regulation of Runx1 in mouse uterus during the peri-implantation period.

    PubMed

    Bai, Zhi-Kun; Li, Dang-Dang; Guo, Chuan-Hui; Yang, Zhan-Qing; Cao, Hang; Guo, Bin; Yue, Zhan-Peng

    2015-10-01

    Runx1 transcription factor is a key developmental regulator. However, little is known about the effects of Runx1 on embryo implantation and decidualization. The aim of this study is to examine the expression and regulation of Runx1 in mouse uterus during the peri-implantation period. There was no evident Runx1 mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy, Runx1 mRNA was mainly localized in the subluminal stroma surrounding the implanting blastocyst. A similar result was observed in the estrogen-activated implantation uterus. Simultaneously, a high level of Runx1 mRNA expression was detected on days 6-8 of pregnancy and under artificial decidualization. 8-Br-cAMP could induce the expression of Runx1 mRNA in the uterine stromal cells. Moreover, the induction was obviously blocked by PKA inhibitor H89. Inhibition of Runx1 with specific siRNA could decrease the proliferation of stromal cells and expression of decidual markers Prl8a2 and Prl3c1 in the uterine stromal cells. Further study found that inhibition of Runx1 could also suppress the expression of Cox-2, mPGES-1 and Mmp2 genes in uterine stromal cells. Estrogen and progesterone could induce the expression of Runx1 mRNA in ovariectomized mouse uterus and uterine stromal cells. Taken together, these data suggest that Runx1 may play an important role during mouse decidualization.

  18. Time course of gene expression during mouse skeletal muscle hypertrophy.

    PubMed

    Chaillou, Thomas; Lee, Jonah D; England, Jonathan H; Esser, Karyn A; McCarthy, John J

    2013-10-01

    The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.

  19. Haemopexin affects iron distribution and ferritin expression in mouse brain

    PubMed Central

    Morello, Noemi; Tonoli, Elisabetta; Logrand, Federica; Fiorito, Veronica; Fagoonee, Sharmila; Turco, Emilia; Silengo, Lorenzo; Vercelli, Alessandro; Altruda, Fiorella; Tolosano, Emanuela

    2009-01-01

    Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases. PMID:19120692

  20. Expression of the Lingo/LERN gene family during mouse embryogenesis.

    PubMed

    Haines, Bryan P; Rigby, Peter W J

    2008-01-01

    We have analysed the expression during mouse development of the four member Lingo/LERN gene family which encodes type 1 transmembrane proteins containing 12 extracellular leucine rich repeats, an immunoglobulin C2 domain and a short intracellular tail. Each family member has a distinct pattern of expression in the mouse embryo as is the case for the related NLRR, FLRT and LRRTM gene families. Lingo1/LERN1 is expressed in the developing trigeminal, facio-acoustic and dorsal root ganglia. An interesting expression pattern is also observed in the somites with expression localising to the inner surface of the dermomyotome in the ventro-caudal lip. Further expression is seen in lateral cells of the hindbrain and midbrain, lateral cells in the motor horn of the neural tube, the otic vesicle epithelium and epithelium associated with the developing gut. Lingo3/LERN2 is expressed in a broad but specific pattern in many tissues across the embryo. Lingo2/LERN3 is seen in a population of cells lying adjacent to the epithelial lining of the olfactory pit while Lingo4/LERN4 is expressed in the neural tube in a subset of progenitors adjacent to the motor neurons. Expression of all Lingo/LERN genes increases as the embryo develops but is low in the adult with only Lingo1/LERN1 and Lingo2/LERN3 being detectable in adult brain.

  1. Analysis of spatial-temporal gene expression patterns reveals dynamics and regionalization in developing mouse brain.

    PubMed

    Chou, Shen-Ju; Wang, Chindi; Sintupisut, Nardnisa; Niou, Zhen-Xian; Lin, Chih-Hsu; Li, Ker-Chau; Yeang, Chen-Hsiang

    2016-01-20

    Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development.

  2. Building a 5-HT3A Receptor Expression Map in the Mouse Brain

    PubMed Central

    Koyama, Yoshihisa; Kondo, Makoto; Shimada, Shoichi

    2017-01-01

    Of the many serotonin receptors, the type 3 receptors (5-HT3R) are the only ionotropic ones, playing a key role in fast synaptic transmission and cognitive and emotional brain function through controlled neuronal excitation. To better understand the various functions of 5-HT3Rs, it is very important to know their expression pattern in the central nervous system (CNS). To date, many distributional studies have shown localized 5-HT3R expression in the brain and spinal cord. However, an accurate pattern of 5-HT3R expression in the CNS remains to be elucidated. To investigate the distribution of 5-HT3R in the mouse brain in detail, we performed immunofluorescent staining using 5-HT3AR-GFP transgenic mice. We found strong 5-HT3AR expression in the olfactory bulb, cerebral cortex, hippocampus, and amygdala; and partial expression in the pons, medulla, and spinal cord. Meanwhile, the thalamus, hypothalamus, and midbrain exhibited a few 5-HT3AR-expressing cells, and no expression was detected in the cerebellum. Further, double-immunostaining using neural markers confirmed that 5-HT3AR is expressed in GABAergic interneurons containing somatostatin or calretinin. In the present study, we built a 5-HT3AR expression map in the mouse brain. Our findings make significant contributions in elucidating the novel functions of 5-HT3R in the CNS. PMID:28276429

  3. Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology

    PubMed Central

    Szibor, Marten; Dhandapani, Praveen K.; Dufour, Eric; Holmström, Kira M.; Zhuang, Yuan; Salwig, Isabelle; Wittig, Ilka; Heidler, Juliana; Gizatullina, Zemfira; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Nandania, Jatin; Velagapudi, Vidya; Wietelmann, Astrid; Rustin, Pierre; Gellerich, Frank N.; Braun, Thomas

    2017-01-01

    ABSTRACT Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo. PMID:28067626

  4. Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology.

    PubMed

    Szibor, Marten; Dhandapani, Praveen K; Dufour, Eric; Holmström, Kira M; Zhuang, Yuan; Salwig, Isabelle; Wittig, Ilka; Heidler, Juliana; Gizatullina, Zemfira; Gainutdinov, Timur; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Nandania, Jatin; Velagapudi, Vidya; Wietelmann, Astrid; Rustin, Pierre; Gellerich, Frank N; Jacobs, Howard T; Braun, Thomas

    2017-02-01

    Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOX(Rosa26) mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOX(Rosa26) mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.

  5. Differential expression of transcriptional repressor snail gene at implantation site in mouse uterus.

    PubMed

    Ma, Xing-Hong; Hu, Shi-Jun; Yu, Hao; Xu, Li-Bin; Yang, Zeng-Ming

    2006-02-01

    The snail superfamily of zinc-finger transcription factors is involved in pronounced cell movements during both embryonic development and tumor progression. This study was to examine snail expression in mouse uterus during early pregnancy and its regulation under pseudopregnancy, delayed implantation, steroid hormone treatment, and artificial decidualization by in situ hybridization and immunohistochemistry. There was a low level of snail mRNA signal and immunostaining in mouse uteri on day 1-4 of pregnancy. When embryo implanted on day 5, both snail mRNA signal and immunostaining were strongly detected in the subluminal stroma immediately surrounding the implanting blastocyst, but not detected in the inter-implantation sites. Under delayed implantation, there was no detectable snail expression. After delayed implantation was terminated by estrogen treatment and embryo implanted, there was a strong level of snail mRNA and immunostaining in the subluminal stroma surrounding the implanting blastocyst, which was similar to that on day 5 of pregnancy. Furthermore, there was no detectable snail expression in mouse uterus on day 5 of pseudopregnancy. From day 6-8 of pregnancy, both snail mRNA signal and immunostaining were detected in the decidua. Our data suggest that snail may play an important role during mouse embryo implantation.

  6. Regional Expression of MTG Genes in the Developing Mouse Central Nervous System

    PubMed Central

    Alishahi, Amin; Koyano-Nakagawa, Naoko; Nakagawa, Yasushi

    2009-01-01

    Myeloid translocation gene (MTG) proteins are transcriptional repressors that are highly conserved across species. We studied the expression of three members of this gene family, MTGR1, MTG8, and MTG16 in developing mouse central nervous system by in situ hybridization. All of these genes are detected as early as embryonic day 11.5. Because these genes are known to be induced by proneural genes during neurogenesis, we analyzed the expression of MTG genes in relation to two proneural genes, Neurog2 (also known as Ngn2 or Neurogenin 2) and Ascl1 (also known as Mash1). While MTGR1 are generally expressed in regions that also express Neurog2, MTG8 and MTG16 expression is associated more tightly with that of Ascl1-expressing neural progenitor cells. These results suggest the possibility that expression of MTG genes is differentially controlled by specific proneural genes during neurogenesis. PMID:19618476

  7. Cocaine-Induced Behavioral Sensitization Is Associated With Changes in the Expression of Endocannabinoid and Glutamatergic Signaling Systems in the Mouse Prefrontal Cortex

    PubMed Central

    Blanco, Eduardo; Pavón, Francisco J.; Palomino, Ana; Luque-Rojas, María Jesús; Serrano, Antonia; Rivera, Patricia; Bilbao, Ainhoa; Alen, Francisco; Vida, Margarita; Suárez, Juan

    2015-01-01

    Background: Endocannabinoids modulate the glutamatergic excitatory transmission by acting as retrograde messengers. A growing body of studies has reported that both signaling systems in the mesocorticolimbic neural circuitry are involved in the neurobiological mechanisms underlying drug addiction. Methods: We investigated whether the expression of both endocannabinoid and glutamatergic systems in the prefrontal cortex (PFC) were altered by an acute and/or repeated cocaine administration schedule that resulted in behavioral sensitization. We measured the protein and mRNA expression of the main endocannabinoid metabolic enzymes and the cannabinoid receptor type 1 (CB1). We also analyzed the mRNA expression of relevant components of the glutamate-signaling system, including glutamate-synthesizing enzymes, metabotropic receptors, and ionotropic receptors. Results: Although acute cocaine (10mg/kg) produced no significant changes in the endocannabinoid-related proteins, repeated cocaine administration (20mg/kg daily) induced a pronounced increase in the CB1 receptor expression. In addition, acute cocaine administration (10mg/kg) in cocaine-sensitized mice (referred to as cocaine priming) induced a selective increase in the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). These protein changes were accompanied by an overall decrease in the ratios of endocannabinoid synthesis/degradation, especially the N-acyl phosphatidylethanolamine phospholipase D/FAAH and diacylglycerol lipase alpha/MAGL ratios. Regarding mRNA expression, while acute cocaine administration produced a decrease in CB1 receptors and N-acyl phosphatidylethanolamine phospholipase D, repeated cocaine treatment enhanced CB1 receptor expression. Cocaine-sensitized mice that were administered priming injections of cocaine mainly displayed an increased FAAH expression. These endocannabinoid changes were associated with modifications in glutamatergic

  8. Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction.

    PubMed

    Chuva de Sousa Lopes, Susana M; Feijen, Alie; Korving, Jeroen; Korchynskyi, Olexander; Larsson, Jonas; Karlsson, Stefan; ten Dijke, Peter; Lyons, Karen M; Goldschmeding, Roel; Doevendans, Pieter; Mummery, Christine L

    2004-11-01

    Connective tissue growth factor (CTGF) is reported to be a target gene of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) in vitro. Its physiological role in angiogenesis and skeletogenesis during mouse development has been described recently. Here, we have mapped expression of CTGF mRNA during mouse heart development, postnatal adult life, and after experimental myocardial infarction. Furthermore, we investigated the relationship between CTGF and the BMP/TGFbeta signaling pathway in particular during heart development in mutant mice. Postnatally, CTGF expression in the heart became restricted to the atrium. Strikingly, 1 week after myocardial infarction, when myocytes have disappeared from the infarct zone, CTGF and TGFbeta expression as well as activated forms of TGFbeta but not BMP, Smad effector proteins are colocalized exclusively in the fibroblasts of the scar tissue, suggesting possible cooperation between CTGF and TGFbeta during the pathological fibrotic response.

  9. Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women

    PubMed Central

    Pelch, Katherine E.; Schroder, Amy L.; Kimball, Paul A.; Sharpe-Timms, Kathy L.; Davis, J. W.; Nagel, Susan C.

    2010-01-01

    Objective To define the altered gene expression profile of endometriotic lesions in a mouse model of surgically-induced endometriosis Design Autologous experimental mouse model. Setting Medical school department. Animals Adult C57Bl6 mice. Intervention(s) Endometriosis was surgically-induced by auto-transplantation of uterine tissue to the intestinal mesentery. Endometriotic lesions and eutopic uteri were recovered at 3 or 29 days post-induction. Main Outcome Measure(s) Altered gene expression was measured in the endometriotic lesion relative to the eutopic uterus by genome wide cDNA microarray analysis and was confirmed by real time RT-PCR for six genes. Relevant categories of altered genes were identified using gene ontology analysis to determine groups of genes enriched for altered expression. Result(s) The expression of 479 and 114 genes was altered in the endometriotic lesion compared to the eutopic uterus at 3 or 29 days post-induction, respectively. Gene ontology enrichment analysis revealed that genes associated with the extracellular matrix, cell adhesions, immune function, cell growth, and angiogenesis were altered in the endometriotic lesion compared to the eutopic uterus. Conclusion(s) Based on gene expression analysis, the mouse model of surgically-induced endometriosis appears to be a good model for studying the pathophysiology and treatment of endometriosis. PMID:19473656

  10. Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization.

    PubMed

    Xu, Jingjie; Li, Mo; Zhang, Lu; Xiong, Hao; Lai, Lidan; Guo, Meijun; Zong, Teng; Zhang, Dalei; Yang, Bei; Wu, Lei; Tang, Min; Kuang, Haibin

    2015-11-01

    The androgen receptor (AR) is a ligand-activated transcription factor that is important for both the male and female reproductive systems. The expression and regulation of AR in the uterine endometrium during early pregnancy and decidualization remain relatively under-investigated, so we sought to immunohistochemically examine the spatiotemporal expression of AR in mouse uteri during the peri-implantation period as well as in response to specific steroid hormones. AR protein was found in the nuclei of uterine stromal cells starting on pregnancy Days 1 and 2, with its abundance increasing on Days 3 and 4. From pregnancy Days 5 to 9, however, the expression of AR markedly declined in stromal zones of uteri. No signal was detected in the decidualized cells surrounding the site of embryo implantation; moreover, no AR immunostaining was observed in decidualized uterine cells in an artificial oil-induced model of decidualization. Progesterone significantly inhibited AR protein expression, whereas estrogen dramatically elevated AR abundance in the stroma of ovariectomized mouse uteri. Taken together, our results are the first to demonstrate that decidualization and progesterone significantly inhibited the AR protein expression in vivo, whereas estrogen increased AR protein levels in the stromal cells of mouse uteri. These responses might be advantageous for the proliferation and differentiation of uterine stroma and for embryo implantation during early pregnancy.

  11. Distinct patterns of expression of the RB gene family in mouse and human retina.

    PubMed

    Spencer, Clarellen; Pajovic, Sanja; Devlin, Hollie; Dinh, Quynh-Dao; Corson, Timothy W; Gallie, Brenda L

    2005-06-01

    Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.

  12. Transcriptional control of transglutaminase 2 expression in mouse apoptotic thymocytes.

    PubMed

    Sándor, Katalin; Daniel, Bence; Kiss, Bea; Kovács, Fruzsina; Szondy, Zsuzsa

    2016-08-01

    Transglutaminase 2 (TGM2) is a ubiquitously expressed multifunctional protein, which participates in various biological processes including thymocyte apoptosis. As a result, the transcriptional regulation of the gene is complex and must depend on the cell type. Previous studies from our laboratory have shown that in dying thymocytes the expression of Tgm2 is induced by external signals derived from engulfing macrophages, such as retinoids, transforming growth factor (TGF)-β and adenosine, the latter triggering the adenylate cyclase signaling pathway. The existence of TGF-β and retinoid responsive elements in the promoter region of Tgm2 has already been reported, but the intergenic regulatory elements participating in the regulation of Tgm2 have not yet been identified. Here we used publicly available results from DNase I hypersensitivity analysis followed by deep sequencing and chromatin immunoprecipitation followed by deep sequencing against CCCTC-binding factor (CTCF), H3K4me3, H3K4me1 and H3K27ac to map a putative regulatory element set for Tgm2 in thymocytes. By measuring eRNA expressions of these putative enhancers in retinoid, rTGF-β or dibutiryl cAMP-exposed thymocytes we determined which of them are functional. By applying ChIP-qPCR against SMAD4, retinoic acid receptor, retinoid X receptor, cAMP response element binding protein, P300 and H3K27ac under the same conditions, we identified two enhancers of Tgm2, which seem to act as integrators of the TGF-β, retinoid and adenylate cyclase signaling pathways in dying thymocytes. Our study describes a novel strategy to identify and characterize the signal-specific functional enhancer set of a gene by integrating genome-wide datasets and measuring the production of enhancer specific RNA molecules.

  13. The expression pattern of Follistatin-like 1 in mouse central nervous system development.

    PubMed

    Yang, Yang; Liu, Junhua; Mao, Huihua; Hu, Yu-An; Yan, Yan; Zhao, Chunjie

    2009-10-01

    Follistatin-like 1 (Fstl1), also named TSC-36 (TGF-beta-stimulated clone 36), was first cloned from the mouse osteoblastic MC3T3-E1 cell line and can be up-regulated by TGF-beta. To better study the function of Fstl1 during the development of the mouse central nervous system (CNS), we examined Fstl1 expression in the developing mouse CNS, in detail, by in situ hybridization. Our results show that Fstl1 is strongly expressed in the telencephalon, diencephalon, brainstem, limbic system and spinal cord. In the telencephalon, Fstl1 positive cells are mainly located in the ventricular zone (VZ) and the subventricular zone (SVZ); a relatively weak signal was observed in layers II and III of the neocortex at postnatal stages. Fstl1 expression is robust in the developing hippocampus and persists to P20. In the developing diencephalon and hindbrain, abundant Fstl1 signals were also detected in nuclei including the medial habenular nucleus, the medial dorsal nucleus, the cochlear nuclei and so on. In addition, a strong expression of Fstl1 was detected in the thalamencephalic signal center, as well as in the olfactory cortex from E14.5 to P0. Meanwhile, Fstl1 was expressed in the septal area and the cingulate gyrus of the limbic system after birth. A high level of expression was also observed in the ventral horn of the spinal cord. These results indicate that Fstl1 may play an important role during CNS development in the mouse.

  14. Synthesis, characterization, stereochemistry and antibacterial activity of N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes

    NASA Astrophysics Data System (ADS)

    Ponnuswamy, S.; Pushpalatha, S.; Akila, A.; Raghuvarman, B.; Aravindhan, S.

    2016-12-01

    Three new N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes 3-5 have been synthesized. The structural characterization and the conformational preferences of the compounds 3-5 have been carried out using IR, 1D and 2D NMR spectral data. The NMR spectral data indicate that the N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes 3-5 prefer to exist in twin-chair conformation with partial flattening at amide nitrogen end. In order to avoid A1,3-strain with coplanar acyl groups, the phenyl groups at the amide nitrogen end are forced to occupy axial orientation. X-ray crystal structure of the N-dichloroacetyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonane 4 also supports the twin-chair conformation in the solid state. Furthermore, the antibacterial activity for the compounds 2-5 has been carried out.

  15. Differential regulation of laminin b1 transgene expression in the neonatal and adult mouse brain.

    PubMed

    Sharif, K A; Baker, H; Gudas, L J

    2004-01-01

    Laminins are the major glycoproteins present in basement membrane, a type of extracellular matrix. We showed that the LAMB1 gene, which encodes the laminin beta1 subunit, is transcriptionally activated by retinoic acid in embryonic stem cells. However, little information is available concerning LAMB1 developmental regulation and spatial expression in the adult mouse brain. In this study we used transgenic mice expressing different lengths of LAMB1 promoter driving beta-galactosidase to investigate developmental and adult transcriptional regulation in the regions of the brain in which the laminin beta1 protein is expressed. CNS expression was not observed in transgenic mice carrying a 1.4LAMB1betagal construct. Mice carrying a 2.5LAMB1betagal construct expressed the LAMB1 transgene, as assayed by X-gal staining, only in the molecular layer of the neonatal cerebellum. In contrast, a 3.9LAMB1betagal transgene showed broad regional expression in the adult mouse brain, including the hippocampus, entorhinal cortex, colliculi, striatum, and substantia nigra. Similar expression patterns were observed for the endogenous laminin beta1 protein and for the 3.9LAMB1betagal transgene, analyzed with an antibody against the beta-galactosidase protein. The 3.9LAMB1betagal transgene expression in the hippocampal tri-synaptic circuit suggests a role for the LAMB1 gene in learning and memory.

  16. GRP78 expression and regulation in the mouse uterus during embryo implantation.

    PubMed

    Lin, PengFei; Jin, YaPing; Lan, XiangLi; Yang, YanZhou; Chen, Fenglei; Wang, Nan; Li, Xiao; Sun, YuJie; Wang, AiHua

    2014-06-01

    The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1-4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6-8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.

  17. The structure and regulation of expression of the mouse growth hormone receptor and binding protein

    SciTech Connect

    Talamantes, F.

    1994-12-31

    The mouse growth hormone receptor (mGHR) and the mouse growth hormone-binding protein (mGHBP) are products of a single gene which are generated alternative splicing. The factors that regulate the expression of mGHR and mGHBP mRNA and protein during pregnancy in the mouse are incompletely understood. During pregnancy in the mouse, there are parallel increases in circulating mouse growth hormone (mGH), liver mGHR, and serum mGHBP. The increase in both hepatic mGHR and serum mGHBP begins on Day 9 of gestation and by late gestation the hepatic mGHR content has increased 8-fold and serum mGHBP has increased 30-fold compared with values in nonpregnant controls. A parallel increase occurs in the steady state levels of liver GHR and GHBP encoding mRNAs. The increase in both messages begins on Day 9 of gestation; however, the GHR mRNA reaches maximum levels by Day 13, while the GHBP mRNA continues to increase until the end of pregnancy. The magnitude of the increase in the GHR-encoding message is 15- to 20-fold between nonpregnant and late pregnant mice, and the magnitude of the increase in the GHBP-encoding message is 30- to 50-fold. Both pituitary mGH and the number of conceptuses influence the receptors and binding protein for mGH during pregnancy. 22 refs.

  18. Activation of farnesoid X receptor induces RECK expression in mouse liver

    SciTech Connect

    Peng, Xiaomin; Wu, Weibin; Zhu, Bo; Sun, Zhichao; Ji, Lingling; Ruan, Yuanyuan; Zhou, Meiling; Zhou, Lei; Gu, Jianxin

    2014-01-03

    Highlights: •RECK is a novel transcriptional target gene of FXR in mouse liver. •The FXR response element is located within the intron 1 of RECK gene. •FXR agonist reverses the down-regulation of RECK in the liver in mouse NASH model. -- Abstract: Farnesoid X receptor (FXR) belongs to the ligand-activated nuclear receptor superfamily, and functions as a transcription factor regulating the transcription of numerous genes involved in bile acid homeostasis, lipoprotein and glucose metabolism. In the present study, we identified RECK, a membrane-anchored inhibitor of matrix metalloproteinases, as a novel target gene of FXR in mouse liver. We found that FXR agonist substantially augmented hepatic RECK mRNA and protein expression in vivo and in vitro. FXR regulated the transcription of RECK through directly binding to FXR response element located within intron 1 of the mouse RECK gene. Moreover, FXR agonist reversed the down-regulation of RECK in the livers from mice fed a methionine and choline deficient diet. In summary, our data suggest that RECK is a novel transcriptional target of FXR in mouse liver, and provide clues to better understanding the function of FXR in liver.

  19. Effect of microgravity on gene expression in mouse brain

    PubMed Central

    Iacobas, Dumitru A.; Iacobas, Sanda; Nicchia, Grazia Paola; Desaphy, Jean Francois; Camerino, Diana Conte; Svelto, Maria; Spray, David C.

    2009-01-01

    Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2-week control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extents, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (cell junction, adhesion, extracellular matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by Gene-MAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes. PMID:18704384

  20. Effect of microgravity on gene expression in mouse brain.

    PubMed

    Frigeri, Antonio; Iacobas, Dumitru A; Iacobas, Sanda; Nicchia, Grazia Paola; Desaphy, Jean Francois; Camerino, Diana Conte; Svelto, Maria; Spray, David C

    2008-11-01

    Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2-week control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extents, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (cell junction, adhesion, extracellular matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes.

  1. Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland.

    PubMed

    Miyano, Yuki; Tahara, Shigeyuki; Sakata, Ichiro; Sakai, Takafumi; Abe, Hiroyuki; Kimura, Shioko; Kurotani, Reiko

    2014-04-01

    Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPβ, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

  2. FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype.

    PubMed

    Arsenault, Jason; Gholizadeh, Shervin; Niibori, Yosuke; Pacey, Laura K; Halder, Sebok K; Koxhioni, Enea; Konno, Ayumu; Hirai, Hirokazu; Hampson, David R

    2016-12-01

    Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector-based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35-115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5-6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative

  3. Regulation of X-linked gene expression during early mouse development by Rlim

    PubMed Central

    Wang, Feng; Shin, JongDae; Shea, Jeremy M; Yu, Jun; Bošković, Ana; Byron, Meg; Zhu, Xiaochun; Shalek, Alex K; Regev, Aviv; Lawrence, Jeanne B; Torres, Eduardo M; Zhu, Lihua J; Rando, Oliver J; Bach, Ingolf

    2016-01-01

    Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos. DOI: http://dx.doi.org/10.7554/eLife.19127.001 PMID:27642011

  4. Regulation of X-linked gene expression during early mouse development by Rlim.

    PubMed

    Wang, Feng; Shin, JongDae; Shea, Jeremy M; Yu, Jun; Bošković, Ana; Byron, Meg; Zhu, Xiaochun; Shalek, Alex K; Regev, Aviv; Lawrence, Jeanne B; Torres, Eduardo M; Zhu, Lihua J; Rando, Oliver J; Bach, Ingolf

    2016-09-19

    Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

  5. Development, structure, and keratin expression in C57BL/6J mouse eccrine glands.

    PubMed

    Taylor, D K; Bubier, J A; Silva, K A; Sundberg, J P

    2012-01-01

    Eccrine sweat glands in the mouse are found only on the footpads and, when mature, resemble human eccrine glands. Eccrine gland anlagen were first apparent at 16.5 days postconception (DPC) in mouse embryos as small accumulations of cells in the mesenchymal tissue beneath the developing epidermis resembling hair follicle placodes. These cells extended into the dermis where significant cell organization, duct development, and evidence of the acrosyringium were observed in 6- to 7-postpartum day (PPD) mice. Mouse-specific keratin 1 (K1) and 10 (K10) expression was confined to the strata spinosum and granulosum. In 16.5 and 18.5 DPC embryos, K14 and K17 were both expressed in the stratum basale and diffusely in the gland anlagen. K5 expression closely mimicked K17 throughout gland development. K6 expression was not observed in the developing glands of the embryo but was apparent in the luminal cell layer of the duct by 6 to 7 PPD. By 21 PPD, the gland apertures appeared as depressions in the surface surrounded by cornified squames, and the footpad surface lacked the organized ridge and crease system seen in human fingers. These data serve as a valuable reference for investigators who use genetically engineered mice for skin research.

  6. Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres.

    PubMed

    Chavez, Shawn L; McElroy, Sohyun L; Bossert, Nancy L; De Jonge, Christopher J; Rodriguez, Maria Vera; Leong, Denise E; Behr, Barry; Westphal, Lynn M; Reijo Pera, Renee A

    2014-09-15

    A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development.

  7. PD-1, PD-L1 and PD-L2 expression in mouse prostate cancer.

    PubMed

    Yang, Shijie; Zhang, Qiuyang; Liu, Sen; Wang, Alun R; You, Zongbing

    2016-01-01

    Programmed cell death protein 1 (PD-1) and its ligands PD-L1 and PD-L2 play critical roles in maintaining an immunosuppressive tumor microenvironment. The purpose of the present study was to assess expression of PD-1, PD-L1, and PD-L2 in mouse prostate tumors. A total of 33 mouse prostate tumors derived from Pten-null mice were examined using immunohistochemical staining for PD-1, PD-L1, and PD-L2. The animals were either with interleukin-17 receptor c (Il-17rc) wild-type or knockout genotype, or fed with regular diet or high-fat diet to 30 weeks of age. We found that Il-17rc wild-type mouse prostate tumors had significantly higher levels of PD-1, PD-L1, and PD-L2 than Il-17rc knockout mouse prostate tumors. High-fat diet-induced obese mice had significantly higher levels of PD-1, PD-L1, and PD-L2 in their prostate tumors than lean mice fed with regular diet. Increased expression of PD-1, PD-L1, and PD-L2 was associated with increased number of invasive prostate tumors formed in the Il-17rc wild-type and obese mice compared to the Il-17rc knockout and lean mice, respectively. Our findings suggest that expression of PD-1, PD-L1, and PD-L2 may enhance development of mouse prostate cancer through creating an immunosuppressive tumor microenvironment.

  8. A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

    PubMed

    Norum, Jens Henrik; Bergström, Åsa; Andersson, Agneta Birgitta; Kuiper, Raoul V; Hoelzl, Maria A; Sørlie, Therese; Toftgård, Rune

    2015-08-15

    LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

  9. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    PubMed

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  10. Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development

    PubMed Central

    Maddox, Dennis M; Condie, Brian G

    2001-01-01

    Background Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter γ-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development. Results Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds. Conclusions Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated. PMID:11178105

  11. Expression and localization of aromatase during fetal mouse testis development.

    PubMed

    Borday, Caroline; Merlet, Jorge; Racine, Chrystèle; Habert, René

    2013-01-01

    Les androgènes et les oestrogènes sont indispensables au développement et aux fonctions du testicule. Le testicule est particulièrement sensible aux perturbateurs endocriniens pendant le développement fœtal et beaucoup de perturbateurs endocriniens agissent en modifiant la balance oestrogènes/androgènes. Physiologiquement, cette balance est régulée par une cascade enzymatique qui convertit irréversiblement les androgènes en oestrogènes. Le composant principal de cette cascade est le cytochrome p450 19A1 (appelé couramment aromatase). Le but de ce travail a été d’étudier l’expression de l’aromatase testiculaire au cours du développement fœtal chez la souris.En utilisant une approche par RT-PCR et par western blot, nous avons montré que l’aromatase est exprimée dès 12,5 jours post-conception (jpc) et que l’expression est maximum à 17,5 jpc. Deux transcripts tronqués ont également été détectés par RT-PCR. La localisation cellulaire de l’aromatase a été étudiée par immunohistologie et par immunomarquage après séparation des cellules testiculaires. Cette enzyme est très fortement exprimée dans les cellules de Leydig fœtales. Elle est également exprimée dans les gonocytes mais plus faiblement et à un niveau variable selon les cellules. En revanche, elle est indétectable dans les cellules de Sertoli.En conclusion, cette étude montre pour la première fois chez la souris que 1) l’aromatase est exprimée dès le début de l’ontogenèse testiculaire, 2) elle est exprimée dans les gonocytes suggérant que ces cellules interviennent dans l’endocrinologie testiculaire et que le rapport oestrogènes/androgènes est plus important dans les gonocytes que dans le liquide interstitiel. En outre, on sait que, chez le fœtus de rat l’aromatase est essentiellement exprimée par les cellules de Sertoli. Nous proposons de prendre en compte cette différence inter-espèces comme un nouveau concept pour comprendre les diff

  12. Expression of Slit and Robo genes in the developing mouse heart.

    PubMed

    Medioni, Caroline; Bertrand, Nicolas; Mesbah, Karim; Hudry, Bruno; Dupays, Laurent; Wolstein, Orit; Washkowitz, Andrew J; Papaioannou, Virginia E; Mohun, Timothy J; Harvey, Richard P; Zaffran, Stéphane

    2010-12-01

    Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.

  13. Expression of Gpr177, a Wnt trafficking regulator, in mouse embryogenesis.

    PubMed

    Yu, Hsiao-Man Ivy; Jin, Ying; Fu, Jiang; Hsu, Wei

    2010-07-01

    Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse ortholog required for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist its subcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, we examine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tissues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulating in the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for proper transportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulation of Wnt is crucial for organogenesis in health and disease.

  14. Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas.

    PubMed

    Kaur, Simran; Norkina, Oxana; Ziemer, Donna; Samuelson, Linda C; De Lisle, Robert C

    2004-08-01

    The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.

  15. Expression analysis of the entire MMP and TIMP gene families during mouse tissue development.

    PubMed

    Nuttall, Robert K; Sampieri, Clara L; Pennington, Caroline J; Gill, Sean E; Schultz, Gilbert A; Edwards, Dylan R

    2004-04-09

    Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane-bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.

  16. Laminar and temporal expression dynamics of coding and noncoding RNAs in the mouse neocortex.

    PubMed

    Fertuzinhos, Sofia; Li, Mingfeng; Kawasawa, Yuka Imamura; Ivic, Vedrana; Franjic, Daniel; Singh, Darshani; Crair, Michael; Sestan, Nenad

    2014-03-13

    The hallmark of the cerebral neocortex is its organization into six layers, each containing a characteristic set of cell types and synaptic connections. The transcriptional events involved in laminar development and function still remain elusive. Here, we employed deep sequencing of mRNA and small RNA species to gain insights into transcriptional differences among layers and their temporal dynamics during postnatal development of the mouse primary somatosensory neocortex. We identify a number of coding and noncoding transcripts with specific spatiotemporal expression and splicing patterns. We also identify signature trajectories and gene coexpression networks associated with distinct biological processes and transcriptional overlap between these processes. Finally, we provide data that allow the study of potential miRNA and mRNA interactions. Overall, this study provides an integrated view of the laminar and temporal expression dynamics of coding and noncoding transcripts in the mouse neocortex and a resource for studies of neurodevelopment and transcriptome.

  17. Sequence and neuronal expression of mouse endothelin-1 cDNA.

    PubMed

    Kurama, M; Ishida, N; Matsui, M; Saida, K; Mitsui, Y

    1996-07-17

    We have isolated and sequenced a cDNA that encodes mouse endothelin-1 (ET-1). The putative protein contains 202 amino acids corresponds to the prepro-form of ET-1. Twenty-one amino acids sequence of the putative mature ET-1 was identical with that of rat, porcine, bovine, and human. In situ hybridization histochemistry indicate that ET-1 mRNA was expressed in several hypothalamic nuclei including the suprachiasmatic nucleus (SCN) in rodent brain.

  18. Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain.

    PubMed

    Evans, Paul R; Lee, Sarah E; Smith, Yoland; Hepler, John R

    2014-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen-activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.

  19. Neuregulin 1 Expression and Electrophysiological Abnormalities in the Neuregulin 1 Transmembrane Domain Heterozygous Mutant Mouse

    PubMed Central

    Frank, Elisabeth; Shaw, Alex; Liu, Shijie; Huang, Xu-Feng; Pinault, Didier; Karl, Tim; O’Brien, Terence J.; Shannon Weickert, Cynthia; Jones, Nigel C.

    2015-01-01

    Background The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile. Methods Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine. Results In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice. Interpretation We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function. PMID

  20. Effect of light on global gene expression in the neuroglobin-deficient mouse retina

    PubMed Central

    ILMJÄRV, STEN; REIMETS, RIIN; HUNDAHL, CHRISTIAN ANSGAR; LUUK, HENDRIK

    2014-01-01

    Several previous studies have raised controversy over the functional role of neuroglobin (Ngb) in the retina. Certain studies indicate a significant impact of Ngb on retinal physiology, whereas others are conflicting. The present is an observational study that tested the effect of Ngb deficiency on gene expression in dark- and light-adapted mouse retinas. Large-scale gene expression profiling was performed using GeneChip® Mouse Exon 1.0 ST arrays and the results were compared to publicly available data sets. The lack of Ngb was found to have a minor effect on the light-induced retinal gene expression response. In addition, there was no increase in the expression of marker genes associated with hypoxia, endoplasmic reticulum-stress and oxidative stress in the Ngb-deficient retina. By contrast, several genes were identified that appeared to be differentially expressed between the genotypes when the effect of light was ignored. The present study indicates that Ngb deficiency does not lead to major alternations in light-dependent gene expression response, but leads to subtle systemic differences of a currently unknown functional significance. PMID:25279145

  1. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  2. Sequence analysis, chromosomal location, and developmental expression of the mouse preproendothelin-1 gene

    SciTech Connect

    Maemura, Koji; Kurihara, Hiroki; Kurihara, Yukiko

    1996-01-15

    Recent studies have designated endothelins (ETs) as morphogenetic factors in embryonic development. In the present study, we cloned and characterized the mouse preproendothelin-1 (preproET-1) gene (Edn1) and examined its expression in reference to development. Edn1 comprises five exons, and the open reading frame encodes the 202-amino-acid preproET-1. The sequences and structural organization of Edn1 are highly homologous to those of other species, especially in the terminal 200-bp sequence of the 3{prime}-noncoding region. Interspecific backcross mapping located Edn1 in the central region of chromosomal 13, where a mouse mutation, congenital hydrocephalus (ch), is also mapped. The highest expression of Edn1 mRNA is detected in the lung in adult mice, whereas Edn1 is predominantly expressed in the epithelium and mesenchyme of the pharyngeal arches and in the endothelium of the large arteries. Edn1 expression and ET-1 peptide levels in the lung progressively increase during the perinatal stage, whereas the expression of Edn3, a gene encoding ET-3, reciprocally decreases. These results suggest that Edn1 expression is developmentally regulated in different tissues and organs in mice in a spatial- and temporal-specific manner. 36 refs., 7 figs.

  3. Restricted development of mouse triploid fetuses with disorganized expression of imprinted genes.

    PubMed

    Yamazaki, Wataru; Takahashi, Masashi; Kawahara, Manabu

    2015-12-01

    Eukaryotic species commonly contain a diploid complement of chromosomes. The diploid state appears to be advantageous for mammals because it enables sexual reproduction and facilitates genetic recombination. Nonetheless, the effects of DNA ploidy on mammalian ontogeny have yet to be understood. The present study shows phenotypic features and expression patterns of imprinted genes in tripronucleate diandric and digynic triploid (DAT and DGT) mouse fetuses on embryonic day 10.5 (E10.5). Measurement of crown-rump length revealed that the length of DGT fetuses (1.87 ± 0.13 mm; mean ± standard error of the mean) was much smaller than that of diploid fetuses (4.81 ± 0.05 mm). However, no significant difference was observed in the crown-rump length between diploid and DAT fetuses (3.86 ± 0.43 mm). In DGT fetuses, the expression level of paternally expressed genes, Igf2, Dlk1, Ndn, and Peg3, remained significantly reduced and that of maternally expressed genes, Igf2r and Grb10, increased. Additionally, in DAT fetuses, the Igf2 mRNA expression level was approximately twice that in diploid fetuses, as expected. These results provide the first demonstration that imprinted genes in mouse triploid fetuses show distinctive expression patterns independent of the number of parental-origin haploid sets. These data suggest that both DNA ploidy and asymmetrical functions of parental genomes separately influence mammalian ontogeny.

  4. Expression of the prion-like protein Shadoo in the developing mouse embryo.

    PubMed

    Young, Rachel; Bouet, Stéphan; Polyte, Jacqueline; Le Guillou, Sandrine; Passet, Bruno; Vilotte, Marthe; Castille, Johan; Beringue, Vincent; Le Provost, Fabienne; Laude, Hubert; Vilotte, Jean-Luc

    2011-12-09

    The prion-like protein Shadoo has been suggested to compensate for the lack of PrP in Prnp-knockout mice, explaining their lack of extreme phenotype. In adult mice, both PrP and Shadoo have shown overlapping expression patterns and shared functions. Their expression in the mouse embryo has also been suggested to be complementary, as invalidation of both genes results in embryonic lethality. The developmental expression profile of PrP has been described from post-implantation stages up until birth. However the spatial expression pattern of Shadoo in the developing mouse embryo is not known. We previously described the expression profile of the prion-like protein Shadoo in adult mice using Sprn reporter mice (Sprn-GFP and Sprn-LacZ). Here we used these mice to describe the developmental expression of Shadoo between 10.5 and 14.5 dpc. The observed pattern in specific embryonic cell lineages and in extra-embryonic tissues is consistent with the previously reported phenotype resulting from its knockdown.

  5. Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites.

    PubMed

    Minami, Takeshi; Miyata, Eriko; Sakamoto, Yamato; Kohama, Azusa; Yamazaki, Hideo; Ichida, Seiji

    2009-06-30

    Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 microM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.

  6. Uterine micro-environment and estrogen-dependent regulation of osteopontin expression in mouse blastocyst.

    PubMed

    Xie, Qing-Zhen; Qi, Qian-Rong; Chen, Ying-Xian; Xu, Wang-Ming; Liu, Qian; Yang, Jing

    2013-07-11

    Embryo implantation is a highly synchronized bioprocess between an activated blastocyst and a receptive uterus. In mice, successful implantation relies on the dynamic interplay of estrogen and progesterone; however, the key mediators downstream of these hormones that act on blastocyst competency and endometrium receptivity acquisition are largely unknown. In this study, we showed that the expression of osteopontin (OPN) in mouse blastocysts is regulated by ovarian estrogen and uterine micro-environment. OPN mRNA is up-regulated in mouse blastocyst on day 4 of pregnancy, which is associated with ovarian estrogen secretion peak. Hormone treatment in vivo demonstrated that OPN expression in a blastocyst is regulated by estrogen through an estrogen receptor (ER). Our results of the delayed and activated implantation model showed that OPN expression is induced after estrogen injection. While estrogen treatment during embryo culture in vitro showed less effect on OPN expression, the tubal ligation model on day 3 of pregnancy confirmed that the regulation of estrogen on OPN expression in blastocyst might, through some specific cytokines, have existed in a uterine micro-environment. Collectively, our study presents that estrogen regulates OPN expression and it may play an important role during embryo implantation by activating blastocyst competence and facilitating the endometrium acceptable for active blastocyst.

  7. Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain

    PubMed Central

    Ye, Xin; Smallwood, Philip; Nathans, Jeremy

    2011-01-01

    The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. PMID:21055480

  8. Spatiotemporal expression of caveolin-1 and EMMPRIN during mouse tooth development.

    PubMed

    Shi, Lu; Li, Lingyun; Wang, Ding; Li, Shu; Chen, Zhi; An, Zhengwen

    2016-06-01

    Caveolin-1 is a scaffolding protein involved in the formation of cholesterol-rich caveolae lipid rafts within the plasma membrane and is capable of collecting signaling molecules into the caveolae and regulating their activity, including extracellular matrix metalloproteinase inducer (EMMPRIN). However, detailed expression patterns of caveolin-1 and EMMPRIN in the developing dental germ are largely unknown. The present study investigated the expression patterns of caveolin-1 and EMMPRIN in the developing mouse tooth germ by immunohistochemistry and real-time polymerase chain reaction. At the bud stage, caveolin-1 expression was initiated in the epithelium bud and mesenchymal cells, while EMMPRIN was weakly expressed at this stage. At the cap stage, caveolin-1 protein was located in the lingual part of the tooth germ; however, EMMPRIN protein was located in the labial part. From the bell stage to 2 days postnatal, caveolin-1 expression was detected in the ameloblasts and cervical loop area; with EMMPRIN expression in the ameloblasts and odontoblasts. Real-time polymerase chain reaction results showed that both caveolin-1 and EMMPRIN mRNA levels increased gradually with progression of developmental stages, and peaked at day two postnatal. The current finding suggests that both caveolin-1 and EMMPRIN take part in mouse tooth development, especially in the differentiation and organization of odontogenic tissues.

  9. Dynamic changes in stanniocalcin gene expression in the mouse uterus during early implantation.

    PubMed

    Stasko, S E; DiMattia, G E; Wagner, G F

    2001-03-28

    Blastocyst implantation is accompanied by dramatic changes in gene expression to facilitate decidualization and remodelling of uterine architecture. Stanniocalcin (STC) is a new mammalian polypeptide hormone with roles in ion transport, reproduction and development. Here we report dynamic changes in STC mRNA and protein distributions in the early post-implantation mouse uterus. In the non-pregnant state, STC gene expression was confined to the uterine lumenal epithelium. Following implantation STC gene expression shifted to mesometrial stromal cells bordering the uterine lumen. Between E6.5-E8.5 expression shifted once more to cells of the mesometrial lateral sinusoids, and then declined thereafter. Intriguingly immunoreactive STC did not entirely co-localize with areas of high STC gene activity and instead appeared to accumulate in presumptive targets of the hormone (uterine epithelium, stromal and decidual cells, trophoblastic giant cells). STC is only the fourth gene identified as being expressed mesometrially in the uterus following implantation.

  10. Estrogen effects on the expression of Brx in the brain and pituitary of the mouse.

    PubMed

    Eddington, David O; Baldwin, Emily L; Segars, James H; Wu, T J

    2006-04-28

    A member of the Dbl family of oncoproteins was discovered in breast cancer tissue extracts. This novel protein, designated Brx, contains an estrogen-receptor binding motif and is highly expressed in hormone-responsive breast tissue. Due to its ability to augment ligand-dependent activation of estrogen receptors, we analyzed the expression of Brx in the adult mouse brain and pituitary. Results indicated that Brx was expressed in specific regions of the brain and pituitary. Furthermore, the results indicate that differences exist in both brain and pituitary tissue of male and female mice with greater expression in the female. However, estrogen did not influence Brx expression in ovariectomized mice. The anatomical studies support a role for Brx in its association with the estrogen receptor and that Brx may be involved in neuronal and pituitary function in a sexually dimorphic manner.

  11. A genome-scale map of expression for a mouse brain section obtained using voxelation

    PubMed Central

    Chin, Mark H.; Geng, Alex B.; Khan, Arshad H.; Qian, Wei-Jun; Petyuk, Vladislav A.; Boline, Jyl; Levy, Shawn; Toga, Arthur W.; Smith, Richard D.; Leahy, Richard M.; Smith, Desmond J.

    2011-01-01

    Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological diseases. We have reconstructed two-dimensional images of gene expression for 20,000 genes in a coronal slice of the mouse brain at the level of the striatum by using microarrays in combination with voxelation at a resolution of 1 mm3. Good reliability of the microarray results were confirmed using multiple replicates, subsequent quantitative RT-PCR voxelation, mass spectrometry voxelation, and publicly available in situ hybridization data. Known and novel genes were identified with expression patterns localized to defined substructures within the brain. In addition, genes with unexpected patterns were identified, and cluster analysis identified a set of genes with a gradient of dorsal/ventral expression not restricted to known anatomical boundaries. The genome-scale maps of gene expression obtained using voxelation will be a valuable tool for the neuroscience community. PMID:17504947

  12. A genome-scale map of expression for a mouse brain section obtained using voxelation

    SciTech Connect

    Chin, Mark H.; Geng, Alex B.; Khan, Arshad H.; Qian, Weijun; Petyuk, Vladislav A.; Boline, Jyl; Levy, Shawn; Toga, Arthur W.; Smith, Richard D.; Leahy, Richard M.; Smith, Desmond J.

    2007-08-20

    Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological diseases. We have reconstructed 2- dimensional images of gene expression for 20,000 genes in a coronal slice of the mouse brain at the level of the striatum by using microarrays in combination with voxelation at a resolution of 1 mm3. Good reliability of the microarray results were confirmed using multiple replicates, subsequent quantitative RT-PCR voxelation, mass spectrometry voxelation and publicly available in situ hybridization data. Known and novel genes were identified with expression patterns localized to defined substructures within the brain. In addition, genes with unexpected patterns were identified and cluster analysis identified a set of genes with a gradient of dorsal/ventral expression not restricted to known anatomical boundaries. The genome-scale maps of gene expression obtained using voxelation will be a valuable tool for the neuroscience community.

  13. Differential expression of axon-sorting molecules in mouse olfactory sensory neurons.

    PubMed

    Ihara, Naoki; Nakashima, Ai; Hoshina, Naosuke; Ikegaya, Yuji; Takeuchi, Haruki

    2016-08-01

    In the mouse olfactory system, the axons of olfactory sensory neurons that express the same type of odorant receptor (OR) converge to a specific set of glomeruli in the olfactory bulb (OB). It is widely accepted that expressed OR molecules instruct glomerular segregation by regulating the expression of axon-sorting molecules. Although the relationship between the expression of axon-sorting molecules and OR types has been analyzed in detail, those between the expressions of axon-sorting molecules remain to be elucidated. Here we collected the expression profiles of four axon-sorting molecules from a large number of glomeruli in the OB. These molecules demonstrated position-independent mosaic expressions, but their patterns were not identical in the OB. Comparing their expressions identified positive and negative correlations between several pairs of genes even though they showed various expressions. Furthermore, the principal component analysis revealed that the factor loadings in the principal component 1, which explain the largest amount of variation, were most likely to reflect the degree of the cyclic nucleotide-gated (CNG) channel dependence on the expression of axon-sorting molecules. Thus, neural activity generated through the CNG channel is a major component in the generation of a wide variety of expressions of axon-sorting molecules in glomerular segregation.

  14. BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos.

    PubMed

    Liu, W; Stein, P; Cheng, X; Yang, W; Shao, N-Y; Morrisey, E E; Schultz, R M; You, J

    2014-12-01

    Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant-negative approach suppresses Nanog expression, and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)), a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.

  15. Prostaglandin F2α upregulates Slit/Robo expression in mouse corpus luteum during luteolysis.

    PubMed

    Zhang, Xuejing; Li, Jianhua; Liu, Jiali; Luo, Haoshu; Gou, Kemian; Cui, Sheng

    2013-09-01

    Prostaglandin F2 α (PGF2 α) is a key factor in the triggering of the regression of the corpus luteum (CL). Furthermore, it has been reported that Slit/Robo signaling is involved in the regulation of luteolysis. However, the interactions between PGF2 α and Slit/Robo in the progression of luteolysis remain to be established. This study was designed to determine whether luteolysis is regulated by the interactions of PGF2 α and Slit/Robo in the mouse CL. Real-time PCR and immunohistochemistry results showed that Slit2 and its receptor Robo1 are highly and specifically co-expressed in the mouse CL. Functional studies showed that Slit/Robo participates in mouse luteolysis by enhancing cell apoptosis and upregulating caspase3 expression. Both in vitro and in vivo studies showed that PGF2 α significantly increases the expression of Slit2 and Robo1 during luteolysis through protein kinase C-dependent ERK1/2 and P38 MAPK signaling pathways, whereas an inhibitor of Slit/Robo signaling significantly decreases the stimulating effect of PGF2 α on luteolysis. These findings indicate that Slit/Robo signaling plays important roles in PGF2 α-induced luteolysis by mediating the PGF2 α signaling pathway in the CL.

  16. Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb.

    PubMed

    Yamanaka, S; Johnson, O N; Norflus, F; Boles, D J; Proia, R L

    1994-06-01

    Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

    PubMed Central

    Fan, Xiaohu; Lang, Haili; Zhou, Xianpei; Zhang, Li; Yin, Rong; Maciejko, Jessica; Giannitsos, Vasiliki; Motyka, Bruce; Medin, Jeffrey A.; Platt, Jeffrey L.

    2010-01-01

    Abstract The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human α-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings. PMID:20163247

  18. Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

    PubMed

    Diaz-Guerra, M; Haddow, S; Bauluz, C; Jorcano, J L; Cano, A; Balmain, A; Quintanilla, M

    1992-02-01

    Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.

  19. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  20. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    SciTech Connect

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  1. Expression of cadherin-8 mRNA in the developing mouse central nervous system.

    PubMed

    Korematsu, K; Redies, C

    1997-10-20

    The expression of cadherin-8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin-8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin-8-positive structures also express this molecule at earlier developmental stages (E12.5-E16). The cadherin-8-positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin-8-positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin-8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin-8-positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin-8 expression in the Purkinje cell layer. In the hindbrain, cadherin-8 is expressed by several cranial nerve nuclei. Results from this study show that cadherin-8 expression in the embryonic and postnatal mouse brain is restricted to specific developing gray matter structures. These data support the idea that cadherins are a family of molecules whose expression provides a molecular code for the regionalization of the developing vertebrate brain.

  2. Differential distribution of cubilin and megalin expression in the mouse embryo.

    PubMed

    Drake, Christopher J; Fleming, Paul A; Larue, Amanda C; Barth, Jeremy L; Chintalapudi, Mastan R; Argraves, W Scott

    2004-03-01

    Cubilin and megalin are cell surface proteins that work cooperatively in many absorptive epithelia to mediate endocytosis of lipoproteins, vitamin carriers, and other proteins. Here we have investigated the coordinate expression of these receptors during mouse development. Our findings indicate that while there are sites where the receptors are co-expressed, there are other tissues where expression is not overlapping. Apical cubilin expression is pronounced in the extraembryonic visceral endoderm (VE) of 6-9.5 days postcoitum (dpc) embryos. By contrast, little megalin expression is evident in the VE at 6 dpc. However, megalin expression in the VE increases as development progresses (7.5-9.5 dpc), although it is not as uniformly distributed as cubilin. Punctate expression of megalin is also apparent in the region of the ectoplacental cone associated with decidual cells, whereas cubilin expression is not seen in association with the ectoplacenta. Strong expression of megalin is observed in the neural ectoderm, neural plate and neural tube (6-8.5 dpc), but cubilin expression is not apparent in any of these tissues. At 8.5 dpc, megalin is expressed in the developing endothelial cells of blood islands, whereas cubilin is absent from these cells. Finally, cubilin, but not megalin, is expressed by a subpopulation of cells dispersed within the 7.5 dpc embryonic endoderm and having a migratory morphology. In summary, the co-expression of cubilin and megalin in the VE is consistent with the two proteins functioning jointly in this tissue. However, the differential distribution pattern indicates that the proteins also function independent of one another. Furthermore, the finding of megalin expression in blood island endothelial cells and cubilin expression in embryonic endoderm highlight potential new developmental roles for these proteins.

  3. Inference of immune cell composition on the expression profiles of mouse tissue

    PubMed Central

    Chen, Ziyi; Huang, Anfei; Sun, Jiya; Jiang, Taijiao; Qin, F. Xiao-Feng; Wu, Aiping

    2017-01-01

    Mice are some of the widely used experimental animal models for studying human diseases. Defining the compositions of immune cell populations in various tissues from experimental mouse models is critical to understanding the involvement of immune responses in various physiological and patho-physiological conditions. However, non-lymphoid tissues are normally composed of vast and diverse cellular components, which make it difficult to quantify the relative proportions of immune cell types. Here we report the development of a computational algorithm, ImmuCC, to infer the relative compositions of 25 immune cell types in mouse tissues using microarray-based mRNA expression data. The ImmuCC algorithm showed good performance and robustness in many simulated datasets. Remarkable concordances were observed when ImmuCC was used on three public datasets, one including enriched immune cells, one with normal single positive T cells, and one with leukemia cell samples. To validate the performance of ImmuCC objectively, thorough cross-comparison of ImmuCC predicted compositions and flow cytometry results was done with in-house generated datasets collected from four distinct mouse lymphoid tissues and three different types of tumor tissues. The good correlation and biologically meaningful results demonstrate the broad utility of ImmuCC for assessing immune cell composition in diverse mouse tissues under various conditions. PMID:28084418

  4. Gene expression based mouse brain parcellation using Markov random field regularized non-negative matrix factorization

    NASA Astrophysics Data System (ADS)

    Pathak, Sayan D.; Haynor, David R.; Thompson, Carol L.; Lein, Ed; Hawrylycz, Michael

    2009-02-01

    Understanding the geography of genetic expression in the mouse brain has opened previously unexplored avenues in neuroinformatics. The Allen Brain Atlas (www.brain-map.org) (ABA) provides genome-wide colorimetric in situ hybridization (ISH) gene expression images at high spatial resolution, all mapped to a common three-dimensional 200μm3 spatial framework defined by the Allen Reference Atlas (ARA) and is a unique data set for studying expression based structural and functional organization of the brain. The goal of this study was to facilitate an unbiased data-driven structural partitioning of the major structures in the mouse brain. We have developed an algorithm that uses nonnegative matrix factorization (NMF) to perform parts based analysis of ISH gene expression images. The standard NMF approach and its variants are limited in their ability to flexibly integrate prior knowledge, in the context of spatial data. In this paper, we introduce spatial connectivity as an additional regularization in NMF decomposition via the use of Markov Random Fields (mNMF). The mNMF algorithm alternates neighborhood updates with iterations of the standard NMF algorithm to exploit spatial correlations in the data. We present the algorithm and show the sub-divisions of hippocampus and somatosensory-cortex obtained via this approach. The results are compared with established neuroanatomic knowledge. We also highlight novel gene expression based sub divisions of the hippocampus identified by using the mNMF algorithm.

  5. Genomic analysis of sexual dimorphism of gene expression in the mouse adrenal gland.

    PubMed

    El Wakil, A; Mari, B; Barhanin, J; Lalli, E

    2013-11-01

    A relevant gender difference exists in adrenal physiology and propensity to disease. In mice, a remarkable sexual dimorphism is present in several components of the hypothalamic-pituitary-adrenal axis, with females displaying higher adrenal weight, plasma ACTH, corticosterone, and aldosterone levels than males. The molecular bases of this sexual dimorphism are little known. We have compared global gene expression profiles in males vs. female mouse adrenal glands and also studied the effect that testosterone treatment and castration have on adrenal gene expression in female vs. male mice, respectively. Our study evidenced a set of 71 genes that are coordinately modulated according to sex and hormonal treatments and represent the core sexually dimorphic expression program in the mouse adrenal gland. Moreover, we show that some genes involved in steroid metabolism have a remarkable sexual dimorphic expression and identify new potential markers for the adrenal X-zone, a transitory cellular layer in the inner adrenal cortex, which spontaneously regresses at puberty in males and during the first pregnancy in females and has an uncertain physiological role. Finally, sexually dimorphic expression of the transcriptional regulators Nr5a1 and Nr0b1 may explain at least in part the differences in adrenal steroidogenesis between sexes.

  6. Integrative analysis of the connectivity and gene expression atlases in the mouse brain.

    PubMed

    Ji, Shuiwang; Fakhry, Ahmed; Deng, Houtao

    2014-01-01

    Brain function is the result of interneuron signal transmission controlled by the fundamental biochemistry of each neuron. The biochemical content of a neuron is in turn determined by spatiotemporal gene expression and regulation encoded into the genomic regulatory networks. It is thus of particular interest to elucidate the relationship between gene expression patterns and connectivity in the brain. However, systematic studies of this relationship in a single mammalian brain are lacking to date. Here, we investigate this relationship in the mouse brain using the Allen Brain Atlas data. We employ computational models for predicting brain connectivity from gene expression data. In addition to giving competitive predictive performance, these models can rank the genes according to their predictive power. We show that gene expression is predictive of connectivity in the mouse brain when the connectivity signals are discretized. When the expression patterns of 4084 genes are used, we obtain a predictive accuracy of 93%. Our results also show that a small number of genes can almost give the full predictive power of using thousands of genes. We can achieve a prediction accuracy of 91% by using only 25 genes. Gene ontology analysis of the highly ranked genes shows that they are enriched for connectivity related processes.

  7. Huddling facilitates expression of daily torpor in the large Japanese field mouse Apodemus speciosus.

    PubMed

    Eto, Takeshi; Sakamoto, Shinsuke H; Okubo, Yoshinobu; Koshimoto, Chihiro; Kashimura, Atsushi; Morita, Tetsuo

    2014-06-22

    Small endotherms employ multiple adaptations to maintain energy balance in winter, including spontaneous daily torpor and simultaneous huddling. The relationships between these adaptations have been discussed in several previous studies, but it has not been well-established if huddling actually affects the expression of torpor in small endotherms. We examine whether and how huddling affects the expression of torpor in the large Japanese field mouse Apodemus speciosus, which is known to become torpid under artificial winter conditions. The mice were found to adjust expression of torpor in response to the number of cage mates. Torpor frequency and minimum torpid body temperature were both significantly elevated when the number of cage mates was increased, but there was no significant change in torpor bout length. Rewarming rate on arousal was lower when the number of cage mates was increased, suggesting reduction in endogenous rewarming due to exogenous passive rewarming. Food consumption per mouse decreased significantly with increasing number of cage mates. Thus, our study demonstrates that social thermoregulatory behaviors such as huddling can facilitate expression of spontaneous daily torpor in small rodents. These findings suggest that energy constraints, such as ambient temperature and food availability may not be the only modulating factors on the expression of daily torpor.

  8. Regulation of retinoid X receptor gamma expression by fed state in mouse liver

    SciTech Connect

    Park, Sangkyu; Lee, Yoo Jeong; Ko, Eun Hee; Kim, Jae-woo

    2015-02-27

    Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting–feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting–feeding cycle. - Highlights: • Refeeding increases the RXRγ expression level in mouse liver. • RXRγ expression is induced by high glucose condition in primary hepatocytes. • RXRγ and LXRα have synergistic effect on SREBP-1c promoter activity. • RXRγ binds to LXRE(-299/-280) located within SREBP-1c promoter region and interacts with LXRα.

  9. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    SciTech Connect

    Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  10. Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.

    PubMed

    Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit

    2017-01-20

    CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

  11. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs.

    PubMed

    Nassenstein, Christina; Kwong, Kevin; Taylor-Clark, Thomas; Kollarik, Marian; Macglashan, Donald M; Braun, Armin; Undem, Bradley J

    2008-03-15

    Transient receptor potential (TRP) A1 and TRPM8 are ion channels that have been localized to afferent nociceptive nerves. These TRP channels may be of particular relevance to respiratory nociceptors in that they can be activated by various inhaled irritants and/or cold air. We addressed the hypothesis that mouse vagal sensory nerves projecting to the airways express TRPA1 and TRPM8 and that they can be activated via these receptors. Single cell RT-PCR analysis revealed that TRPA1 mRNA, but not TRPM8, is uniformly expressed in lung-labelled TRPV1-expressing vagal sensory neurons. Neither TRPA1 nor TRPM8 mRNA was expressed in TRPV1-negative neurons. Capsaicin-sensitive, but not capsaicin-insensitive, lung-specific neurons responded to cinnamaldehyde, a TRPA1 agonist, with increases in intracellular calcium. Menthol, a TRPM8 agonist, was ineffective at increasing cellular calcium in lung-specific vagal sensory neurons. Cinnamaldehyde also induced TRPA1-like inward currents (as measured by means of whole cell patch clamp recordings) in capsaicin-sensitive neurons. In an ex vivo vagal innervated mouse lung preparation, cinnamaldehyde evoked action potential discharge in mouse vagal C-fibres with a peak frequency similar to that observed with capsaicin. Cinnamaldehyde inhalation in vivo mimicked capsaicin in eliciting strong central-reflex changes in breathing pattern. Taken together, our results support the hypothesis that TRPA1, but not TRPM8, is expressed in vagal sensory nerves innervating the airways. TRPA1 activation provides a mechanism by which certain environmental stimuli may elicit action potential discharge in airway afferent C-fibres and the consequent nocifensor reflexes.

  12. Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains

    PubMed Central

    Szabo, R.; Samson, A. L.; Lawrence, D. A.; Medcalf, R. L.; Bugge, T. H.

    2017-01-01

    Summary Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density SNP analysis, bioinformatics, and genome editing was used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat−/− mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel “passenger mutation”-free isogenic C57BL/6J-Plat−/− and FVB/NJ-Plat−/− mouse strains by introducing an 11 bp deletion in the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes. PMID:27079292

  13. [Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

    PubMed

    Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

    2010-01-01

    Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

  14. Anti-cancer agents based on N-acyl-2, 3-dihydro-1H-pyrrolo[2,3-b] quinoline derivatives and a method of making

    DOEpatents

    Gakh, Andrei; Krasavin, Mikhail; Karapetian, Ruben; Rufanov, Konstantin A; Konstantinov, Igor; Godovykh, Elena; Soldatkina, Olga; Sosnov, Andrey V

    2013-04-16

    The present disclosure relates to novel compounds that can be used as anti-cancer agents in the prostate cancer therapy. In particular, the invention relates to N-acyl derivatives of 2,3-dihydro-1H-pyrrolo[2,3-b]quinolines having the structural Formula (I), ##STR00001## stereoisomers, tautomers, racemics, prodrugs, metabolites thereof, or pharmaceutically acceptable salt and/or solvate thereof. The meaning of R1 is independently selected from H; C1-C6 Alkyl, cyclo-Alkyl or iso-Alkyl substituents; R2 is selected from C1-C6 Alkyl, cyclo-Alkyl or iso-Alkyl; substituted or non-substituted, fused or non-fused to substituted or non-substituted aromatic ring, aryl or heteroaryl groups. The invention also relates to methods for preparing said compounds, and to pharmaceutical compositions comprising said compounds.

  15. Biochemical engineering of the N-acyl side chain of sialic acids alters the kinetics of a glycosylated potassium channel Kv3.1.

    PubMed

    Hall, M Kristen; Reutter, Werner; Lindhorst, Thisbe; Schwalbe, Ruth A

    2011-10-20

    The sialic acid of complex N-glycans can be biochemically engineered by substituting the physiological precursor N-acetylmannosamine with non-natural N-acylmannosamines. The Kv3.1 glycoprotein, a neuronal voltage-gated potassium channel, contains sialic acid. Western blots of the Kv3.1 glycoprotein isolated from transfected B35 neuroblastoma cells incubated with N-acylmannosamines verified sialylated N-glycans attached to the Kv3.1 glycoprotein. Outward ionic currents of Kv3.1 transfected B35 cells treated with N-pentanoylmannosamine or N-propanoylmannosamine had slower activation and inactivation rates than those of untreated cells. Therefore, the N-acyl side chain of sialic acid is intimately connected with the activation and inactivation rates of this glycosylated potassium channel.

  16. Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry.

    PubMed Central

    Aplin, R T; Baldwin, J E; Roach, P L; Robinson, C V; Schofield, C J

    1993-01-01

    Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. Images Figure 1 PMID:8396910

  17. Synthesis, characterization stereochemistry and anti-bacterial evaluation of certain N-acyl-c-3,t-3-dimethyl-r-2,c-6-diphenylpiperidin-4-ones

    NASA Astrophysics Data System (ADS)

    Ponnuswamy, S.; Kayalvizhi, R.; Jamesh, M.; Uma Maheswari, J.; Thenmozhi, M.; Ponnuswamy, M. N.

    2016-09-01

    A new series of N-acyl-c-3,t-3-dimethyl-r-2,c-6-diphenylpiperidin-4-ones 2-6 has been synthesized and characterized using IR, mass, 1H, 13C, DEPT and 2D (COSY and HSQC) NMR spectral techniques. The NMR spectral data indicate that the N-acylpiperidin-4-ones 2-6 prefer to exist in a distorted boat conformation B1 with coplanar orientation of N-C=O moiety. The stereodynamics of these systems have been studied by recording the dynamic 1H NMR spectra of compound 4, and the energy barrier for N-CO rotation is determined to be 52.75 kJ/mol. Furthermore the compounds 1-5 show significant antibacterial activity.

  18. Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines.

    PubMed

    Sato, Yasuto; Fukuyama, Yuko; Nonami, Hiroshi; Erra-Balsells, Rosa; Stortz, Carlos A; Cerezo, Alberto S; Matulewicz, María C

    2007-12-10

    Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.

  19. Development of an extraction method and LC-MS analysis for N-acylated-l-homoserine lactones (AHLs) in wastewater treatment biofilms.

    PubMed

    Wang, Jinfeng; Ding, Lili; Li, Kan; Schmieder, Wilhelm; Geng, Jinju; Xu, Ke; Zhang, Yan; Ren, Hongqiang

    2017-01-15

    N-Acylated-l-homoserine lactones (AHLs) play a vital role in Gram-negative bacteria communication by promoting the formation of extracellular polymeric substances (EPS) and biofilms. However, the low concentration of these AHL signals makes the process difficult to understand. A robust and sensitive pretreatment method for AHL detection was developed in this work. Compared with eight different solid-phase extraction (SPE) columns and three various solid extraction method, we found that the UE (ultrasonic extraction) and an Oasis hydrophilic-lipophilic-balanced (HLB) sorbent in column format combined with ultra-performance liquid chromatography linked to tandem mass spectrometry (UPLC-MS/MS) can be successfully used for systematic pretreating moving bed biofilm reactor (MBBR) biological samples to extract AHLs and determine concentration of AHLs in wastewater treatment biofilms. This easy-to-follow protocol makes it ideal for quantitative analyses of AHLs in wastewater treatment biofilms.

  20. Heterologous expression of the adenosine A1 receptor in transgenic mouse retina.

    PubMed

    Li, Ning; Salom, David; Zhang, Li; Harris, Tim; Ballesteros, Juan A; Golczak, Marcin; Jastrzebska, Beata; Palczewski, Krzysztof; Kurahara, Carole; Juan, Todd; Jordan, Steven; Salon, John A

    2007-07-17

    Traditional cell-based systems used to express integral membrane receptors have yet to produce protein samples of sufficient quality for structural study. Herein we report an in vivo method that harnesses the photoreceptor system of the retina to heterologously express G protein-coupled receptors in a biochemically homogeneous and pharmacologically functional conformation. As an example we show that the adenosine A1 receptor, when placed under the influence of the mouse opsin promoter and rhodopsin rod outer segment targeting sequence, localized to the photoreceptor cells of transgenic retina. The resulting receptor protein was uniformly glycosylated and pharmacologically well behaved. By comparison, we demonstrated in a control experiment that opsin, when expressed in the liver, had a complex pattern of glycosylation. Upon solubilization, the retinal adenosine A1 receptor retained binding characteristics similar to its starting material. This expression method may prove generally useful for generating high-quality G protein-coupled receptors for structural studies.

  1. Gene expression analysis of mouse embryonic stem cells following levitation in an ultrasound standing wave trap.

    PubMed

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-02-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine.

  2. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation.

    PubMed

    Saga, Y; Hata, N; Koseki, H; Taketo, M M

    1997-07-15

    We isolated a novel bHLH protein gene Mesp2 (for mesoderm posterior 2) that cross-hybridizes with Mesp1 expressed in the early mouse mesoderm. Mesp2 is expressed in the rostral presomitic mesoderm, but down-regulated immediately after the formation of the segmented somites. To determine the function of MesP2 protein (MesP2) in somitogenesis, we generated Mesp2-deficient mice by gene targeting. The homozygous Mesp2 (-/-) mice died shortly after birth and had fused vertebral columns and dorsal root ganglia, with impaired sclerotomal polarity. The earliest defect in the homozygous embryos was a lack of segmented somites. Their disruption of the metameric features, altered expression of Mox-1, Pax-1, and Dll1, and lack of expression of Notch1, Notch2, and FGFR1 suggested that MesP2 controls sclerotomal polarity by regulating the signaling systems mediated by notch-delta and FGF, which are essential for segmentation.

  3. Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression.

    PubMed

    Dockery, Lauren; Gerfen, Jennifer; Harview, Christina; Rahn-Lee, Charlotte; Horton, Rachel; Park, Yaena; Davis, Tamara L

    2009-05-16

    A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.

  4. Expression of macro non-coding RNAs Meg8 and Irm in mouse embryonic development.

    PubMed

    Gu, Tiantian; He, Hongjuan; Han, Zhengbin; Zeng, Tiebo; Huang, Zhijun; Liu, Qi; Gu, Ning; Chen, Yan; Sugimoto, Kenkichi; Jiang, Huijie; Wu, Qiong

    2012-07-01

    Non-coding RNAs (ncRNAs) Meg8 and Irm were previously identified as alternatively splicing isoforms of Rian gene. Ascertaining ncRNAs spatiotemporal expression patterns is crucial for understanding the physiological roles of ncRNAs during tissue and organ development. In this study in mouse embryos, we focused on the developmental regulation expression of imprinted macro ncRNAs, Meg8 and Irm by using in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). The in situ hybridization results showed that Meg8 and Irm were expressed in the developing brain at embryonic day 10.5 (E10.5) and E11.5, while Irm expression signals were strikingly detected in the somite, where Meg8 expression signals were undetectable. By E15.5, they were expressed in brain, tongue, liver, lung and neuroendocrine tissues, while Irm displayed more restricted expression in tongue and skeletal muscle than Meg8. Furthermore, quantitative analysis confirmed that they were highly expressed in tongue and brain at E12.5, E15.5 and E18.5. These results indicated that Meg8 and Irm might be coordinately expressed and functionally correlated in diverse of organs. Notably, Irm was more closely associated with morphogenesis of skeletal muscle in contrast to Meg8 during embryonic development.

  5. Expression profiling of uniparental mouse embryos is inefficient in identifying novel imprinted genes.

    PubMed

    Ruf, Nico; Dünzinger, Ulrich; Brinckmann, Anja; Haaf, Thomas; Nürnberg, Peter; Zechner, Ulrich

    2006-04-01

    Imprinted genes are expressed from only one allele in a parent-of-origin-specific manner. We here describe a systematic approach to identify novel imprinted genes using quantification of allele-specific expression by Pyrosequencing, a highly accurate method to detect allele-specific expression differences. Sixty-eight candidate imprinted transcripts mapping to known imprinted chromosomal regions were selected from a recent expression profiling study of uniparental mouse embryos and analyzed. Three novel imprinted transcripts encoding putative non-protein-coding RNAs were identified on the basis of parent-of-origin-specific monoallelic expression in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross embryos. In addition, four transcripts with preferential expression of a strain-specific allele were found. Intriguingly, a vast majority of the analyzed transcripts showed no imprinting-associated expression in F1 embryos. These data strengthen the view that a large fraction of nonimprinted genes is differentially expressed between parthenogenetic and androgenetic embryos and question the efficiency of expression profiling of uniparental embryos to identify novel imprinted genes.

  6. Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver.

    PubMed

    Argyros, Orestis; Wong, Suet Ping; Fedonidis, Constantinos; Tolmachov, Oleg; Waddington, Simon N; Howe, Steven J; Niceta, Marcello; Coutelle, Charles; Harbottle, Richard P

    2011-05-01

    We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

  7. Identification and developmental analysis of endothelin receptor type-A expressing cells in the mouse kidney.

    PubMed

    Kitazawa, Taro; Sato, Takahiro; Nishiyama, Koichi; Asai, Rieko; Arima, Yuichiro; Uchijima, Yasunobu; Kurihara, Yukiko; Kurihara, Hiroki

    2011-10-01

    The endothelin (Edn) system plays pleiotropic roles in renal function and various disease processes through two distinct G protein-coupled receptors, Edn receptors type-A (Ednra) and type-B (Ednrb). However, difficulties in the accurate identification of receptor-expressing cells in situ have made it difficult to dissect their diverse action in renal (patho)physiology. We have recently established mouse lines in which lacZ and EGFP are 'knocked-in' to the Ednra locus to faithfully mark Ednra-expressing cells. Here we analyzed these mice for their expression in the kidney to characterize Ednra-expressing cells. Ednra expression was first observed in undifferentiated mesenchymal cells around the ureteric bud at E12.5. Thereafter, Ednra expression was widely observed in vascular smooth muscle cells, JG cells and mesenchymal cells in the interstitium. After growth, the expression became confined to vascular smooth muscle cells, pericytes and renin-producing JG cells. By contrast, most cells in the nephron and vascular endothelial cells did not express Ednra. These results indicate that Ednra expression may be linked with non-epithelial fate determination and differentiation of metanephric mesenchyme. Ednra-lacZ/EGFP knock-in mice may serve as a useful tool in studies on renal function and pathophysiology of various renal diseases.

  8. Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

    SciTech Connect

    Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan; Surh, Young-Joon

    2013-09-06

    Highlights: •Thymoquinone inhibits phorbol ester-induced COX-2 expression in mouse skin. •Thymoquinone attenuates phosphorylation of IκBα and DNA binding of NF-κB in mouse skin. •Thymoquinone inhibits phosphorylation of p38 MAP kinase, JNK and Akt in mouse skin. •Thymoquinone induces the expression of cytoprotective proteins in mouse skin. -- Abstract: Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

  9. Mining and characterization of ubiquitin E3 ligases expressed in the mouse testis

    PubMed Central

    2012-01-01

    Background Ubiquitin-mediated protein modification and degradation are believed to play important roles in mammalian spermatogenesis. The catalogues of ubiquitin activating enzymes, conjugating enzymes, and ligases (E3s) have been known for mammals such as mice and humans. However, a systematic characterization of E3s expressed during spermatogenesis has not been carried out. Results In present study, we set out to mine E3s from the mouse genome and to characterize their expression pattern, subcellular localization, and enzymatic activities based on microarray data and biochemical assays. We identified 398 putative E3s belonging to the RING, U-box, and HECT subfamilies and found that most genes were conserved between mice and humans. We discovered that 73 of them were highly or specifically expressed in the testes based on the microarray expression data. We selected 10 putative E3 genes to examine their mRNA expression pattern, and several genes to study their subcellular localization and E3 ligase activity. RT-PCR results showed that all the selected genes were predominately expressed in the testis. Some putative E3s were localized in the cytoplasm while others were in both the cytoplasm and the nucleus. Moreover, all the selected proteins were enzymatically active as demonstrated by in vitro and in vivo assays. Conclusions We have identified a large number of putative E3s that are expressed during mouse spermatogenesis. Among these, a significant portion is highly or specifically expressed in the testis. Subcellular localization and enzymatic activity assays suggested that these E3s might execute diverse functions in mammalian spermatogenesis. Our results may serve as an initial guide to the field for further functional analysis. PMID:22992278

  10. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells

    PubMed Central

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-01-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  11. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages.

    PubMed

    Cao, Heping; Cao, Fangping; Roussel, Anne-Marie; Anderson, Richard A

    2013-12-01

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.

  12. Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

    PubMed Central

    Gstir, Ronald; Schafferer, Simon; Scheideler, Marcel; Misslinger, Matthias; Griehl, Matthias; Daschil, Nina; Humpel, Christian; Obermair, Gerald J.; Schmuckermair, Claudia; Striessnig, Joerg; Flucher, Bernhard E.

    2014-01-01

    We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson's disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer's disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer's disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs. PMID:25344396

  13. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    PubMed

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  14. Increased expression of miRNA-146a in Alzheimer’s disease transgenic mouse models

    PubMed Central

    Li, Y.Y.; Cui, J.G.; Hill, J.M.; Bhattacharjee, S.; Zhao, Y.; Lukiw, W.J.

    2017-01-01

    A mouse and human brain-enriched micro-RNA-146a (miRNA-146a) is known to be important in modulating the innate immune response and inflammatory signaling in certain immunological and brain cell types. In this study we examined miRNA-146a levels in early-, moderate- and late-stage Alzheimer’s disease (AD) neocortex and hippocampus, in several human primary brain and retinal cell lines, and in 5 different transgenic mouse models of AD including Tg2576, TgCRND8, PSAPP, 3xTg-AD and 5xFAD. Inducible expression of miRNA-146a was found to be significantly up-regulated in a primary co-culture of human neuronal–glial (HNG) cells stressed using interleukin1-beta (IL-1β), and this up-regulation was quenched using specific NF-κB inhibitors including curcumin. Expression of miRNA-146a correlated with senile plaque density and synaptic pathology in Tg2576 and in 5xFAD transgenic mouse models used in the study of this common neurodegenerative disorder. PMID:20934487

  15. miR-29 regulates Tet1 expression and contributes to early differentiation of mouse ESCs

    PubMed Central

    Yang, Dehua; Li, Song; Le, Weidong

    2016-01-01

    The ten-eleven translocation-1 (Tet1), which converts 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC), plays important roles in many important biological processes, such as mouse embryonic stem cells (ESCs) maintenance. However, the mechanisms for Tet-1 regulation remain largely unknown. Here we showed that miR-29 family (miR-29a, miR-29b and miR-29c) can directly repress Tet1 expression. We found that Tet1 was highly expressed and 5hmC was presented at relatively high levels in mouse ESCs, but the levels of both Tet1 and 5hmC were reduced during the early differentiation of ESCs. On the contrary, miR-29 level was increased in this process. ESCs stably transfecting with miR-29 precursors showed lower levels of Tet1 protein and 5hmC. Furthermore, we demonstrated that miR-29 overexpression selectively affected cell lineage markers and skewed ESC differentiation, which was similar in Tet1 knockdown ESCs. Our results indicate that miR-29 is a direct regulator of Tet1 in mouse ESCs. PMID:27449105

  16. Monitoring Long Interspersed Nuclear Element 1 Expression During Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Bodak, Maxime; Ciaudo, Constance

    2016-01-01

    Long Interspersed Elements-1 (LINE-1 or L1) are a class of transposable elements which account for almost 19 % of the mouse genome. This represents around 600,000 L1 fragments, among which it is estimated that 3000 intact copies still remain capable to retrotranspose and to generate deleterious mutation by insertion into genomic coding region. In differentiated cells, full length L1 are transcriptionally repressed by DNA methylation. However at the blastocyst stage, L1 elements are subject to a demethylation wave and able to be expressed and to be inserted into new genomic locations. Mouse Embryonic Stem Cells (mESCs) are pluripotent stem cells derived from the inner cell mass of blastocysts. Mouse ESCs can be maintained undifferentiated under controlled culture conditions or induced into the three primary germ layers, therefore they represent a suitable model to follow mechanisms involved in L1 repression during the process of differentiation of mESCs. This protocol presents how to maintain culture of undifferentiated mESCs, induce their differentiation, and monitor L1 expression at the transcriptional and translational levels. L1 transcriptional levels are assessed by real-time qRT-PCR performed on total RNA extracts using specific L1 primers and translation levels are measured by Western blot analysis of L1 protein ORF1 using a specific L1 antibody.

  17. Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons

    PubMed Central

    Molet, Jenny; Gunn, Benjamin G.; Ressler, Kerry

    2015-01-01

    Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools. PMID:26402844

  18. Electric Pulse Stimulation of Cultured Murine Muscle Cells Reproduces Gene Expression Changes of Trained Mouse Muscle

    PubMed Central

    Burch, Nathalie; Arnold, Anne-Sophie; Item, Flurin; Summermatter, Serge; Brochmann Santana Santos, Gesa; Christe, Martine; Boutellier, Urs; Toigo, Marco; Handschin, Christoph

    2010-01-01

    Adequate levels of physical activity are at the center of a healthy lifestyle. However, the molecular mechanisms that mediate the beneficial effects of exercise remain enigmatic. This gap in knowledge is caused by the lack of an amenable experimental model system. Therefore, we optimized electric pulse stimulation of muscle cells to closely recapitulate the plastic changes in gene expression observed in a trained skeletal muscle. The exact experimental conditions were established using the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) as a marker for an endurance-trained muscle fiber. We subsequently compared the changes in the relative expression of metabolic and myofibrillar genes in the muscle cell system with those observed in mouse muscle in vivo following either an acute or repeated bouts of treadmill exercise. Importantly, in electrically stimulated C2C12 mouse muscle cells, the qualitative transcriptional adaptations were almost identical to those in trained muscle, but differ from the acute effects of exercise on muscle gene expression. In addition, significant alterations in the expression of myofibrillar proteins indicate that this stimulation could be used to modulate the fiber-type of muscle cells in culture. Our data thus describe an experimental cell culture model for the study of at least some of the transcriptional aspects of skeletal muscle adaptation to physical activity. This system will be useful for the study of the molecular mechanisms that regulate exercise adaptation in muscle. PMID:20532042

  19. Expression of the ankyrin repeat domain 6 gene (Ankrd6) during mouse brain development.

    PubMed

    Tissir, F; Bar, I; Goffinet, A M; Lambert De Rouvroit, C

    2002-08-01

    The structure and developmental expression pattern of the ankyrin repeat domain 6 (Ankrd6) gene, initially named Diversin, were studied in the mouse. Ankrd6 is transcribed as a 5.8-kb mRNA composed of 15 exons that encodes a 712 amino acid protein with 6 ankyrin repeats. Ankrd6 is expressed prominently in the developing brain from E12 to maturity, suggesting a role during brain development. In embryos, expression is maximal in ventricular zones of neuronal proliferation and intermediate zones of neuronal migration and extends to postmigratory neuronal fields during the postnatal period. In the mature brain, the Ankrd6-related signal is highest in cortical layer II, granule cells of the dentate gyrus, olfactory granules and a subset of Purkinje cells in the vestibulocerebellum. Ankrd6 is related to the Drosophila gene Diego, which interacts with Flamingo in the regulation of planar cell polarity (Feiguin et al., 2001). However, the canvas of Ankrd6 expression does not match closely that of the three mouse Flamingo homologs, Celsr1-3 (Tissir et al., 2002). These data suggest that Ankrd6 may be involved in brain development in interaction with Celsr/Flamingo but also other signaling pathways.

  20. Continued Discovery of Transcriptional Units Expressed in Cells of the Mouse Mononuclear Phagocyte Lineage

    PubMed Central

    Wells, Christine A.; Ravasi, Timothy; Sultana, Razvan; Yagi, Ken; Carninci, Piero; Bono, Hidemasa; Faulkner, Geoffrey; Okazaki, Yasushi; Quackenbush, John; Hume, David A.; Lyons, Paul A.

    2003-01-01

    The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome. PMID:12819134

  1. Expression Profiling of the Solute Carrier Gene Family in the Mouse BrainS⃞

    PubMed Central

    Dahlin, Amber; Royall, Josh; Hohmann, John G.; Wang, Joanne

    2009-01-01

    The solute carrier (Slc) superfamily is a major group of membrane transport proteins present in mammalian cells. Although Slc transporters play essential and diverse roles in the central nervous system, the localization and function of the vast majority of Slc genes in the mammalian brain are largely unknown. Using high-throughput in situ hybridization data generated by the Allen Brain Atlas, we systematically and quantitatively analyzed the spatial and cellular distribution of 307 Slc genes, which represent nearly 90% of presently known mouse Slc genes, in the adult C57BL/6J mouse brain. Our analysis showed that 252 (82%) of the 307 Slc genes are present in the brain, and a large proportion of these genes were detected at low to moderate expression levels. Evaluation of 20 anatomical brain subdivisions demonstrated a comparable level of Slc gene complexity but significant difference in transcript enrichment. The distribution of the expressed Slc genes was diverse, ranging from near-ubiquitous to highly localized. Functional annotation in 20 brain regions, including the blood-brain and blood-cerebral spinal fluid (CSF) barriers, suggests major roles of Slc transporters in supporting brain energy utilization, neurotransmission, nutrient supply, and CSF production. Furthermore, hierarchical cluster analysis revealed intricate Slc expression patterns associated with neuroanatomical organization. Our studies also revealed Slc genes present within defined brain microstructures and described the putative cell types expressing individual Slc genes. These results provide a useful resource for investigators to explore the roles of Slc genes in neurophysiological and pathological processes. PMID:19179540

  2. HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human.

    PubMed Central

    Bedford, F K; Ashworth, A; Enver, T; Wiedemann, L M

    1993-01-01

    We describe the cloning of a novel homeodomain-containing gene, which is highly conserved between mouse and human. The human cDNA was initially isolated from human haematopoietic tissue and denoted HEX (haematopoietically expressed homeobox). Sequence analysis of the coding sequences from mouse and the partial cDNA from human shows that the homeodomain is most closely related to those of the HIx and HOX11 proteins. The HEX gene is present as a single copy in the human genome. Analysis of murine genomic DNA shows, in addition to an intron-containing gene homologous to HEX, the presence of a processed copy of the gene which has arisen within the last few million years. Analysis of human and murine haematopoietic cells and cell lines, revealed expression of the HEX gene in multipotential progenitors, as well as cells of the B-lymphocyte and myeloid lineages. However HEX was not expressed in T-lymphocytes or erythroid cells. This pattern of HEX gene expression suggests that it may play a role in haematopoietic differentiation. Images PMID:8096636

  3. Analyses of Allele-Specific Gene Expression in Highly Divergent Mouse Crosses Identifies Pervasive Allelic Imbalance

    PubMed Central

    Crowley, James J; Zhabotynsky, Vasyl; Sun, Wei; Huang, Shunping; Pakatci, Isa Kemal; Kim, Yunjung; Wang, Jeremy R; Morgan, Andrew P; Calaway, John D; Aylor, David L; Yun, Zaining; Bell, Timothy A; Buus, Ryan J; Calaway, Mark E; Didion, John P; Gooch, Terry J; Hansen, Stephanie D; Robinson, Nashiya N; Shaw, Ginger D; Spence, Jason S; Quackenbush, Corey R; Barrick, Cordelia J; Nonneman, Randal J.; Kim, Kyungsu; Xenakis, James; Xie, Yuying; Valdar, William; Lenarcic, Alan B; Wang, Wei; Welsh, Catherine E; Fu, Chen-Ping; Zhang, Zhaojun; Holt, James; Guo, Zhishan; Threadgill, David W; Tarantino, Lisa M; Miller, Darla R; Zou, Fei; McMillan, Leonard; Sullivan, Patrick F; de Villena, Fernando Pardo-Manuel

    2015-01-01

    Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Since regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in this process. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. These effects influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a novel, global allelic imbalance in favor of the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals. PMID:25730764

  4. Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

    PubMed Central

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-01-01

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning. PMID:24786090

  5. Expression of chondrogenic potential of mouse trunk neural crest cells by FGF2 treatment.

    PubMed

    Ido, Atsushi; Ito, Kazuo

    2006-02-01

    There is a significant difference between the developmental patterns of cranial and trunk neural crest cells in the amniote. Thus, whereas cranial neural crest cells generate bone and cartilage, trunk neural crest cells do not contribute to skeletal derivatives. We examined whether mouse trunk neural crest cells can undergo chondrogenesis to analyze how the difference between the developmental patterns of cranial and trunk neural crest cells arises. Our present data demonstrate that mouse trunk neural crest cells have chondrogenic potential and that fibroblast growth factor (FGF) 2 is an inducing factor for their chondrogenesis in vitro. FGF2 altered the expression patterns of Hox9 genes and Id2, a cranial neural crest cell marker. These results suggest that environmental cues may play essential roles in generating the difference between developmental patterns of cranial and trunk neural crest cells.

  6. Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

    PubMed Central

    Mills, J S; Needham, M; Parker, M G

    1987-01-01

    A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein. Images PMID:3502715

  7. Proteomic study of differential protein expression in mouse lung tissues after aerosolized ricin poisoning.

    PubMed

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-04-28

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

  8. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    PubMed Central

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE. Methods We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE. Results We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier. Conclusion These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular

  9. Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

    PubMed Central

    Dobie, K W; Lee, M; Fantes, J A; Graham, E; Clark, A J; Springbett, A; Lathe, R; McClenaghan, M

    1996-01-01

    Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event. Images Fig. 3 Fig. 4 PMID:8692874

  10. A Novel Cytoplasmic Tail Motif Regulates Mouse Corin Expression on the Cell Surface

    PubMed Central

    Li, Hui; Zhang, Yue; Wang, Lina; Dong, Ningzheng; Qi, Xiaofei; Wu, Qingyu

    2015-01-01

    Type II transmembrane serine proteases (TTSPs) are important in many biological processes. Cell surface expression is critical for TTSP activation and function. To date, the mechanism underlying TTSP cell surface expression is poorly understood. Corin is a TTSP and acts as the pro-atrial natriuretic peptide convertase that is essential for sodium homeostasis and normal blood pressure. In this study, we investigated how cytoplasmic tail sequences may regulate corin expression and activation on the cell surface. By site-directed mutagenesis, we made mouse corin proteins with truncations or point-mutations in the cytoplasmic tail. We expressed the mutants in transfected HEK293 cells and analyzed corin cell surface expression and activation by Western blotting and flow cytometry. We found that corin truncation mutants lacking a Lys-Phe-Gln sequence at residues 71–73 had higher levels of cell surface expression and activation compared with that in wild-type corin. When Lys-71, Phe-72 and Gln-73 residues were mutated together, but not individually, in corin with the full-length cytoplasmic tail, increased levels of cell surface expression and zymogen activation were also observed. These results indicate that residues Lys-71, Phe-72 and Gln-73 serve as a novel retention motif in the intracellular pathway to regulate corin cell surface expression and activation. PMID:26241673

  11. Expression of the Astrocyte Water Channel Aquaporin-4 in the Mouse Brain.

    PubMed

    Hubbard, Jacqueline A; Hsu, Mike S; Seldin, Marcus M; Binder, Devin K

    2015-01-01

    Aquaporin-4 (AQP4) is a bidirectional water channel that is found on astrocytes throughout the central nervous system. Expression is particularly high around areas in contact with cerebrospinal fluid, suggesting that AQP4 plays a role in fluid exchange between the cerebrospinal fluid compartments and the brain. Despite its significant role in the brain, the overall spatial and region-specific distribution of AQP4 has yet to be fully characterized. In this study, we used Western blotting and immunohistochemical techniques to characterize AQP4 expression and localization throughout the mouse brain. We observed AQP4 expression throughout the forebrain, subcortical areas, and brainstem. AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus. We found that AQP4 immunoreactivity was profuse on glial cells bordering ventricles, blood vessels, and subarachnoid space. Throughout the brain, AQP4 was expressed on astrocytic end-feet surrounding blood vessels but was also heterogeneously expressed in brain tissue parenchyma and neuropil, often with striking laminar specificity. In the cerebellum, we showed that AQP4 colocalized with the proteoglycan brevican, which is synthesized by and expressed on cerebellar astrocytes. Despite the high abundance of AQP4 in the cerebellum, its functional significance has yet to be investigated. Given the known role of AQP4 in synaptic plasticity in the hippocampus, the widespread and region-specific expression pattern of AQP4 suggests involvement not only in fluid balance and ion homeostasis but also local synaptic plasticity and function in distinct brain circuits.

  12. Expression analysis of G Protein-Coupled Receptors in mouse macrophages

    PubMed Central

    Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

    2008-01-01

    Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery. PMID:18442421

  13. Mouse oocytes suppress miR-322-5p expression in ovarian granulosa cells

    PubMed Central

    SUMITOMO, Jun-ichi; EMORI, Chihiro; MATSUNO, Yuta; UENO, Mizuki; KAWASAKI, Kurenai; ENDO, Takaho A.; SHIROGUCHI, Katsuyuki; FUJII, Wataru; NAITO, Kunihiko; SUGIURA, Koji

    2016-01-01

    This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15–/–/Gdf9+/– (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice. Taken together, these results show that ODPFs suppress the expression of miR-322 in cumulus cells. Gene ontology analysis of putative miR-322 targets whose expression was detected in MGCs with RNA-sequencing suggested that multiple biological processes are affected by miR-322 in MGCs. These results demonstrate that ODPFs regulate miRNA expression in granulosa cells and that this regulation may participate in the differential control of cumulus cell versus MGC functions. Therefore, the ODPF-mediated regulation of cumulus cells takes place at both transcriptional and post-transcriptional levels. PMID:27180925

  14. Cerebral endothelial expression of Robo1 affects brain infiltration of polymorphonuclear neutrophils during mouse stroke recovery.

    PubMed

    Gangaraju, Sandhya; Sultan, Khadeejah; Whitehead, Shawn N; Nilchi, Ladan; Slinn, Jacqueline; Li, Xuesheng; Hou, Sheng T

    2013-06-01

    Increased brain infiltration of polymorphonuclear neutrophils (PMNs) occurs early after stroke and is important in eliciting brain inflammatory response during stroke recovery. In order to understand the molecular mechanism of PMN entry, we investigated the expression and requirement for Slit1, a chemorepulsive guidance cue, and its cognate receptor, Robo1, in a long-term recovery mouse model of cerebral ischemia. The expression levels of Robo1 were significantly decreased bilaterally at 24h following reperfusion. Robo1 expression levels remained suppressed in the ipsilateral cortex until 28d post MCAO-reperfusion, while the levels of Robo1 in the contralateral cortex recovered to the level of sham-operated mouse by 7d reperfusion. Circulating PMNs express high levels of Slit1, but not Robo1. Influx of PMNs into the ischemic core area occurred early (24h) after cerebral ischemia, when endothelial Robo1 expression was significantly reduced in the ischemic brain, indicating that Robo1 may form a repulsive barrier to PMN entry into the brain parenchyma. Indeed, blocking Slit1 on PMNs in a transwell migration assay in combination with an antibody blocking of Robo1 on human umbilical vein endothelial cells (HUVEC) significantly increased PMN transmigration during oxygen glucose deprivation, an in vitro model of ischemia. Collectively, in the normal brain, the presence of Slit1 on PMNs, and Robo1 on cerebral endothelial cells, generated a repulsive force to prevent the infiltration of PMNs into the brain. During stroke recovery, a transient reduction in Robo1 expression on the cerebral endothelial cells allowed the uncontrolled infiltration of Slit1-expressing PMNs into the brain causing inflammatory reactions.

  15. Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia.

    PubMed

    Schrans-Stassen, B H; van de Kant, H J; de Rooij, D G; van Pelt, A M

    1999-12-01

    The proto-oncogene c-kit is encoded at the white-spotting locus and in the mouse mutations at this locus affect the precursor cells of melanocytes, hematopoietic cells, and germ cells. c-kit is expressed in type A spermatogonia, but whether or not c-kit is present both in undifferentiated and differentiating type A spermatogonia or only in the latter cell type is still a matter of debate. Using the vitamin A-deficient mouse model, we studied messenger RNA (mRNA) and protein expression in undifferentiated and differentiating type A spermatogonia. Furthermore, we quantified the immuno-positive type A spermatogonia in the epithelial stages VI, VII, IX/X, and XII in normal mice to correlate c-kit expression in type A spermatogonia with the differentiation of these cells. Our results show that in the VAD situation undifferentiated type A spermatogonia express little c-kit mRNA. The A spermatogonia with a larger nucleus expressed c-Kit protein, whereas the A spermatogonia with a smaller one did not. After induction of differentiation of these cells into type A1 spermatogonia, c-kit mRNA was enhanced. The percentage of A spermatogonia expressing c-Kit protein did not change during this process, suggesting that A spermatogonia, which are committed to differentiate express c-kit. Under normal circumstances in epithelial stage VI 16%+/-2% (mean +/- SD), in VII 45%+/-15%, in IX/X 78%+/-14% and in XII 90%+/-1.9% of the type A spermatogonia were c-kit positive, suggesting that Aaligned spermatogonia gradually change from c-Kit negative to c-Kit positive cells before their differentiation into A1 spermatogonia. It is concluded that c-kit can be used as a marker for differentiation of undifferentiated into differentiating type A spermatogonia.

  16. FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype

    PubMed Central

    Arsenault, Jason; Gholizadeh, Shervin; Niibori, Yosuke; Pacey, Laura K.; Halder, Sebok K.; Koxhioni, Enea; Konno, Ayumu; Hirai, Hirokazu; Hampson, David R.

    2016-01-01

    Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector–based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35–115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5–6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long

  17. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    SciTech Connect

    Xu, W.; Desnick, R.J.; Kozak, C.A.

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  18. Expression of Variant Ribosomal RNA Genes in Mouse Oocytes and Preimplantation Embryos1

    PubMed Central

    Ihara, Motomasa; Tseng, Hung; Schultz, Richard M.

    2011-01-01

    Ribosomal DNA (rDNA) is not composed of multiple copies of identical transcription units, as commonly believed, but rather of at least seven rDNA variant subtypes that are expressed in somatic cells. This finding raises the possibility that ribosome function may be modulated as proposed by the ribosome filter hypothesis. We report here that mouse oocytes and preimplantation embryos express all the rDNA variants except variant V and that there is no marked developmental change in the qualitative pattern of variant expression. The maternal and embryonic ribosome pools are therefore quite similar, minimizing the likelihood that developmental changes in composition of the ribosome population are critical for preimplantation development. PMID:21209414

  19. Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-1.

    PubMed

    Campbell, C; Stanley, P

    1983-11-01

    Two rare and dominant mutants of Chinese hamster ovary (CHO) cells, LEC11 and LEC12, express the mouse embryonic antigen SSEA-1. Parental CHO cells and the revertants, LEC11.R9 and LEC12.R10, do not express this antigen as detected by a sensitive radioimmunoassay with a monoclonal antibody to SSEA-1. The presence of the SSEA-1 determinant correlates with the apparent de novo expression of specific N-acetylglucosaminide alpha(1,3)fucosyltransferase activities not detected in parental or revertant cell extracts. Several differences in the enzymes substrate specificities and their products have been identified. The combined data suggest that LEC11 and LEC12 mutants result from regulatory mutations affecting different fucosyltransferase genes.

  20. Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy

    PubMed Central

    2010-01-01

    Background The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. Results To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. Conclusions Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation. PMID:20630075

  1. Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos.

    PubMed

    Hirasawa, Ryutaro; Sasaki, Hiroyuki

    2009-01-01

    The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

  2. Gli1 is not required for Pdgfralpha expression during mouse embryonic development.

    PubMed

    Zhang, Xiao-Qun; Afink, Gijs B; Hu, Xin-Rong; Forsberg-Nilsson, Karin; Nistér, Monica

    2005-03-01

    Pdgfra is expressed in the mesenchyme of multiple organs during embryonic development and Pdgfralpha is involved in cell proliferation, differentiation, migration, and apoptosis in many tissues. A fine-tuned regulation of gene transcription is required to achieve these effects. To investigate if the Shh signaling pathway is involved in the tightly regulated Pdgfra expression during embryogenesis, we systematically compared Gli1 and Pdgfralpha mRNA expression patterns in vivo from mouse embryonic day 9.5 to 14.5. We found that an initial partly overlapping expression of Gli1 and Pdgfralpha in the mesenchyme of foregut and somites was changed to different expression patterns when the mesenchyme differentiated into specialized structures such as intestinal villi and chondrocytes. Gli1 and Pdgfra were also expressed differently in the developing lung, heart, central nervous system, skin, tooth, and eye. Importantly, neither Pdgfralpha mRNA patterns nor levels were altered in Ihh mutant embryos although Gli1 and Ptc mRNA levels were dramatically reduced. Our results demonstrate that Gli1 is not required to induce Pdgfra expression during embryonic bone development, and are consistent with previous findings that Pdgfralpha and Hh pathways serve different functions in, e.g., bone, gut, and lung development. However, we cannot exclude the possibility that Glis can have more complex regulatory effects on Pdgfra gene activity, nor can we exclude such effects in pathological conditions.

  3. Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

    PubMed Central

    Ross, S R; Hsu, C L; Choi, Y; Mok, E; Dudley, J P

    1990-01-01

    Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene. Images PMID:1700274

  4. Expression patterns of ubiquitin conjugating enzyme UbcM2 during mouse embryonic development.

    PubMed

    Yanjiang, Xing; Hongjuan, He; Tiantian, Gu; Yan, Zhang; Zhijun, Huang; Qiong, Wu

    2012-01-01

    Ubiquitin conjugating enzyme UbcM2 (Ubiquitin-conjugating enzymes from Mice, the number reveals the identification order) has been implicated in many critical processes, such like growth-inhibiting, mediating cell proliferation and regulation of some transcription factor, but the expression profile during mouse embryo development remains unclear. Hereby, during mid-later embryonic stage, the expression patterns of UbcM2 were examined using in situ hybridization and quantitative real-time PCR (qRT-PCR). The signals were significantly intense in central nervous system and skeletal system, weak in tongue, heart, lung, liver, and kidney. In the central nervous system, UbcM2 was principally expressed in thalamus, external germinal layer of cerebellum (EGL), mitral cell layer of olfactory bulb, hippocampus, marginal zone and ventricular zone of cerebral cortex, and spinal cord. In the skeletal system, UbcM2 was primarily expressed in proliferating cartilage. Furthermore, qRT-PCR analysis displayed that the expression of UbcM2 was ubiquitous at E15.5, most prominent in brain, weaker in lung liver and kidney, accompanied by the lowest level in tongue and heart. During brain development, the expression level of UbcM2 first ascended and then decreased from E12.5 to E18.5, the peak of which sustained starting at E14.5 until E16.5. Together, these results suggest that UbcM2 may play potential roles in the development of mouse diverse tissues and organs, particularly in the development of brain and skeleton.

  5. Expression of cyclins E1 and E2 during mouse development and in neoplasia

    PubMed Central

    Geng, Yan; Yu, Qunyan; Whoriskey, Wendy; Dick, Fred; Tsai, Kenneth Y.; Ford, Heide L.; Biswas, Debajit K.; Pardee, Arthur B.; Amati, Bruno; Jacks, Tyler; Richardson, Andrea; Dyson, Nicholas; Sicinski, Piotr

    2001-01-01

    Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G1/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in ≈24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry. PMID:11687642

  6. Lung arginase expression and activity is increased in cystic fibrosis mouse models.

    PubMed

    Jaecklin, Thomas; Duerr, Julia; Huang, Hailu; Rafii, Mahroukh; Bear, Christine E; Ratjen, Felix; Pencharz, Paul; Kavanagh, Brian P; Mall, Marcus A; Grasemann, Hartmut

    2014-08-01

    The activity of arginase is increased in airway secretions of patients with cystic fibrosis (CF). Downstream products of arginase activity may contribute to CF lung disease. We hypothesized that pulmonary arginase expression and activity would be increased in mouse models of CF and disproportionally increased in CF mice with Pseudomonas aeruginosa pneumonia. Expression of arginase isoforms in lung tissue was quantified with reverse transcriptase-PCR in naive cystic fibrosis transmembrane conductance regulator (Cftr)-deficient mice and β-epithelial sodium channel-overexpressing [β-ENaC-transgenic (Tg)] mice. An isolated lung stable isotope perfusion model was used to measure arginase activity in Cftr-deficient mice before and after intratracheal instillation of Pseudomonas aeruginosa. The expression of arginase-2 in lung was increased in adult Cftr-deficient animals and in newborn β-ENaC-Tg. Arginase-1 lung expression was normal in Cftr-deficient and in newborn β-ENaC-Tg mice, but was increased in β-ENaC-Tg mice at age 1, 3, and 6 wk. Arginase activity was significantly higher in lung (5.0 ± 0.7 vs. 3.2 ± 0.3 nmol·(-1)·h(-1), P = 0.016) and airways (204.6 ± 49.8 vs. 79.3 ± 17.2 nmol·(-1)·h(-1), P = 0.045) of naive Cftr-deficient mice compared with sex-matched wild-type littermate controls. Infection with Pseudomonas aeruginosa resulted in a far greater increase in lung arginase activity in Cftr-deficient mice (10-fold) than in wild-type controls (6-fold) (P = 0.01). This is the first ex vivo characterization of arginase expression and activity in CF mouse lung and airways. Our data show that pulmonary arginase expression and activity is increased in CF mice, especially with Pseudomonas aeruginosa infections.

  7. Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis.

    PubMed

    Sun, Rongfang; Qi, Huayu

    2014-01-01

    Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here

  8. High Dub3 expression in mouse ESCs couples the G1/S checkpoint to pluripotency.

    PubMed

    van der Laan, Siem; Tsanov, Nikolay; Crozet, Carole; Maiorano, Domenico

    2013-11-07

    The molecular mechanism underlying G1/S checkpoint bypass in mouse embryonic stem cells (ESCs) remains unknown. DNA damage blocks S phase entry by inhibiting the CDK2 kinase through destruction of its activator, the Cdc25A phosphatase. We observed high Cdc25A levels in G1 that persist even after DNA damage in mouse ESCs. We also found higher expression of Dub3, a deubiquitylase that controls Cdc25A protein abundance. Moreover, we demonstrate that the Dub3 gene is a direct target of Esrrb, a key transcription factor of the self-renewal machinery. We show that Dub3 expression is strongly downregulated during neural conversion and precedes Cdc25A destabilization, while forced Dub3 expression in ESCs becomes lethal upon differentiation, concomitant to cell-cycle remodeling and lineage commitment. Finally, knockdown of either Dub3 or Cdc25A induced spontaneous differentiation of ESCs. Altogether, these findings couple the self-renewal machinery to cell-cycle control through a deubiquitylase in ESCs.

  9. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response.

    PubMed

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William; Evans, Jodi F

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases.

  10. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes.

    PubMed Central

    Peng, H; Armentano, D; MacKenzie-Graham, L; Shen, R F; Darlington, G; Ledley, F D; Woo, S L

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human alpha 1-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 1-antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes. Images PMID:3186716

  11. The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenic Xenopus laevis.

    PubMed

    Lim, Wayland; Neff, Eric S; Furlow, J David

    2004-06-17

    Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

  12. Cloning of the genes encoding mouse cardiac and skeletal calsequestrins: expression pattern during embryogenesis.

    PubMed

    Park, K W; Goo, J H; Chung, H S; Kim, H; Kim, D H; Park, W J

    1998-09-14

    Calsequestrin is a low-affinity and high-capacity calcium-binding protein in the sarcoplasmic reticulum (SR). In the present study, we have cloned and sequenced mouse cardiac and skeletal calsequestrin cDNAs. The deduced amino acid sequences are highly homologous to those of other mammalian calsequestrins. As expected, the cardiac and skeletal calsequestrins are expressed specifically and exclusively in adult heart and skeletal muscles, respectively. In-situ hybridization was performed to examine the expression pattern of the calsequestrins in the developing mouse and rat embryos. During early organogenesis, the cardiac and skeletal calsequestrin transcripts were detected exclusively in the heart primordium and the myotome of somites, respectively. The cardiac calsequestrin transcripts were later detected in fetal heart and skeletal muscles, whereas the skeletal calsequestrin transcripts were only found in fetal skeletal muscles. These data suggest that the cardiac calsequestrin plays a role in the differentiation and function of heart, and in the function of fetal skeletal muscles in conjunction with the skeletal calsequestrin, but not in the early differentiation of the myotome of somites. The expression of the skeletal calsequestrin in the myotome is regulated probably by myogenin, a myogenic regulatory gene.

  13. Zonal induction of mixed lineage kinase ZPK/DLK/MUK gene expression in regenerating mouse liver.

    PubMed

    Douziech, M; Grondin, G; Loranger, A; Marceau, N; Blouin, R

    1998-08-28

    ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

  14. Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

    PubMed Central

    Saiz, Nestor; Kang, Minjung; Schrode, Nadine; Lou, Xinghua; Hadjantonakis, Anna-Katerina

    2016-01-01

    This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.). PMID:26967230

  15. Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Wu, Honglu

    Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

  16. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response

    PubMed Central

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases. PMID:28191017

  17. Expression and activity of L-Myc in normal mouse development.

    PubMed Central

    Hatton, K S; Mahon, K; Chin, L; Chiu, F C; Lee, H W; Peng, D; Morgenbesser, S D; Horner, J; DePinho, R A

    1996-01-01

    To determine the role of L-Myc in normal mammalian development and its functional relationship to other members of the Myc family, we determined the normal patterns of L-myc gene expression in the developing mouse by RNA in situ hybridization and assessed the phenotypic impact of L-Myc deficiency produced through standard gene targeting methodology. L-myc transcripts were detected in the developing kidney and lung as well as in both the proliferative and the differentiative zones of the brain and neural tube. Despite significant expression of L-myc in developing mouse tissue, homozygous null L-myc mice were found to be viable, reproductively competent, and represented in expected frequencies from heterozygous matings. A detailed histological survey of embryonic and adult tissues, characterization of an embryonic neuronal marker, and measurement of cellular proliferation in situ did not reveal any congenital abnormalities. The lack of an apparent phenotype associated with L-Myc deficiency indicates that L-Myc is dispensable for gross morphological development and argues against a unique role for L-Myc in early central nervous system development as had been previously suggested. Although overlapping expression patterns among myc family members raise the possibility of complementation of L-Myc deficiency by other Myc oncoproteins, compensatory changes in the levels of c- and/or N-myc transcripts were not detected in homozygous null L-myc mice. PMID:8657155

  18. Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation.

    PubMed

    Wu, Weiwei; Kong, Shuangbo; Wang, Bingyan; Chen, Yongjie; Wang, Haibin

    2016-02-01

    Successful implantation requires intimate interactions between a competent blastocyst and a receptive uterus. We recently demonstrated that the aberrant activation of opioid signaling by exogenous ligands adversely affects preimplantation embryonic development and subsequent implantation in mice. However, the underlying machinery governing the dynamic homeostasis of the endogenous opioid system in the uterus during early pregnancy remains elusive. We now show that all three major endogenous opioid precursors are spatiotemporally expressed in the uterus during early pregnancy. Moreover, we observe the well-coordinated expression of the synthetic enzyme prohormone convertases 1/3 (PC1/3) at lower levels and of its inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor (Pcsk1n) and the degrading enzyme membrane metallo-endopeptidase (MME) at higher levels in the receptive uterus. Both estrogen and progestin tend to reduce the uterine levels of opioid ligand precursors in the ovariectomized mouse model. This tight regulation of the endogenous opioid system by PC1/3, Pcsk1n and MME has been further confirmed in physiologically related pseudopregnancy and delayed implantation mouse models. The coordinated regulation of opioid precursor biosynthesis and metabolism helps to create appropriate opioid signaling ensuring uterine receptivity for implantation. Thus, endogenous uterine opioid levels are primarily determined by the coordinated expressions of PC1/3, Pcsk1n and MME under the influence of ovarian progestin and estrogen. Our findings raise an additional cautionary note regarding the effects of opioid abuse on early pregnancy events.

  19. Distinct spatiotemporal expression of ISM1 during mouse and chick development

    PubMed Central

    Osório, Liliana; Wu, Xuewei; Zhou, Zhongjun

    2014-01-01

    Isthmin 1 (ISM1) constitutes the founder of a new family of secreted proteins characterized by the presence of 2 functional domains: thrombospondin type 1 repeat (TSR1) and adhesion-associated domain in MUC4 and other proteins (AMOP). ISM1 was identified in the frog embryo as a member of the FGF8 synexpression group due to its expression in the brain midbrain–hindbrain boundary (MHB) or isthmus. In zebrafish, ISM1 was described as a WNT- and NODAL-regulated gene. The function of ISM1 remains largely elusive. So far, ISM1 has been described as an angiogenesis inhibitor that has a dual function in endothelial cell survival and cell death. For a better understanding of ISM1 function, we examined its spatiotemporal distribution in mouse and chick using RT-PCR, ISH, and IHC analyses. In the mouse, ISM1 transcripts are found in tissues such as the anterior mesendoderm, paraxial and lateral plate mesoderm, MHB and trunk neural tube, as well as in the somites and dermomyotome. In the newborn and adult, ISM1 is prominently expressed in the lung and brain. In addition to its putative role during embryonic and postnatal development, ISM1 may also be important for organ homeostasis in the adult. In the chick embryo, ISM1 transcripts are strongly detected in the ear, eye, and spinal cord primordia. Remarkable differences in ISM1 spatiotemporal expression were found during mouse and chick development, despite the high homology of ISM1 orthologs in these species. PMID:24675886

  20. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    PubMed

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  1. Generation of a transgenic mouse line for conditional expression of human IL-6

    PubMed Central

    Mori, Taiki; Murasawa, Yusuke; Ikai, Rina; Hayakawa, Tomoko; Nakamura, Hiroyuki; Ogiso, Noboru; Niida, Shumpei; Watanabe, Ken

    2016-01-01

    IL-6 is a cytokine that is involved in various physiological and pathological conditions, and approaches using gain-of-function transgenic animals have contributed in elucidating IL-6 function. However, studies of the multiple functions of IL-6 in vivo are very time consuming because they require the generation of transgenic mice that harbor the gene encoding IL-6 under the control of specific promoters to mimic different pathologies. Here, we report the establishment of a conditional human IL-6 transgenic mouse, LGL-IL6, which conditionally expresses human IL-6 by taking advantage of the well-characterized Cre recombinase drivers. PMID:27349442

  2. Orthotopic mouse models of tumor metastasis expressing fluorescent reporters produce imageable circulating tumor cells.

    PubMed

    Hoffman, Robert M

    2014-12-01

    Circulating tumor cells (CTC) are of high importance, since they are potential metastatic precursors and are readily available for prognostic analysis and treatment testing. In this review, we demonstrate the great power that green fluorescent protein (GFP) labeling and orthotopic mouse models of cancer confer to the study of CTCs for isolation and characterization, including metastatic testing in mice and the chick embryo as well as drug response testing in vitro. We also describe a facile method to label patient CTCs ex vivo using a telomerase-expressing GFP-containing adenovirus that will allow the CTC studies described in this review to be translated clinically.

  3. Reduction of Photo Bleaching and Long Term Archiving of Chemically Cleared GFP-Expressing Mouse Brains

    PubMed Central

    Becker, Klaus; Hahn, Christian Markus; Saghafi, Saiedeh; Jährling, Nina; Wanis, Martina; Dodt, Hans-Ulrich

    2014-01-01

    Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years. PMID:25463047

  4. Molecular cloning, expression, and characterization of mouse amine N-sulfotransferases

    SciTech Connect

    Takahashi, Saki |; Sakakibara, Yoichi | Mishiro, Emi |; Kouriki, Haruna; Nobe, Rika |; Kurogi, Katsuhisa; Yasuda, Shin |; Liu, M.-C.; Suiko, Masahito |

    2008-10-31

    By searching the GenBank database, we recently identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (IMAGE Clone ID 679629) and a novel mouse SULT gene (LOC 215895). Sequence analysis revealed that both mouse SULTs belong to the cytosolic SULT3 gene family. The recombinant form of these two newly identified SULTs, designated SULT3A1 and SULT3A2, were expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Both purified SULT3A1 and SULT3A2 exhibited strong amine N-sulfonating activities toward 1-naphthylamine among a variety of endogenous and xenobiotic compounds tested as substrates. Kinetic constants of the sulfation of 1-naphthylamine and 1-naphthol by these two enzymes were determined. Collectively, these results imply that these two amine-sulfonating SULT3s may play essential roles in the metabolism and detoxification of aromatic amine compounds in the body.

  5. Expression of dog microdystrophin in mouse and dog muscles by gene therapy.

    PubMed

    Pichavant, Christophe; Chapdelaine, Pierre; Cerri, Daniel G; Dominique, Jean-Christophe; Quenneville, Simon P; Skuk, Daniel; Kornegay, Joe N; Bizario, João Cs; Xiao, Xiao; Tremblay, Jacques P

    2010-05-01

    Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.

  6. Mapping the dynamic expression of Wnt11 and the lineage contribution of Wnt11-expressing cells during early mouse development

    PubMed Central

    Sinha, Tanvi; Lin, Lizhu; Li, Ding; Davis, Jennifer; Evans, Sylvia; Wynshaw-Boris, Anthony; Wang, Jianbo

    2015-01-01

    Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11-CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11-CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11-CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11-CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained

  7. Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.

    PubMed

    Ikeda, Yayoi; Matsunaga, Yuko; Takiguchi, Masahito; Ikeda, Masa-Aki

    2011-01-01

    Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.

  8. Expression of the deubiquitinating enzyme mUBPy in the mouse brain.

    PubMed

    Bruzzone, Federica; Vallarino, Mauro; Berruti, Giovanna; Angelini, Cristiano

    2008-02-21

    Mouse UBPy (mUBPy) is an ubiquitin-specific protease which belongs to a family of deubiquitinating enzymes (DUBs) implicated in several cellular processes related to both cell growth and differentiation. Previously, Northern blot analysis revealed an important expression of mUBPy in the testis and brain. However, a more comprehensive map of mUBPy localization in the central nervous system (CNS) is still lacking. In this study, we mapped the distribution of mUBPy in the mouse brain using nonradioactive in situ hybridization and immunohistochemical techniques. In general, transcript and protein showed a similar and widespread distribution. In particular, mUBPy was strongly expressed in the hippocampal formation, septal region, ventral pallidum, preoptic nucleus, periventricular nucleus of hypothalamus, compact part of the substantia nigra, ventral tegmental area, cochlear nucleus and granular cell layer of cerebellum. A moderate expression of mUBPy was found in the amygdaloid complex, supraoptic nucleus, arcuate and ventromedial nuclei of hypothalamus, lateral hypothalamic area and lateral and reticular part of the substantia nigra. Double labelling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is generally expressed in neurons and, in specific regions, also in oligodendrocytes. Moreover, by using antisera to TH and mUBPy we found that mUBPy is localized in dopaminergic neurons. The different distribution of mUBPy in the distinct regions of the brain suggests that it could be related to different deubiquitinating processes; in particular, in the areas where it is expressed at high levels, mUBPy could exert a specialized function through its interaction with specific protein substrates.

  9. Expression Pattern of Thyroid Hormone Transporters in the Postnatal Mouse Brain

    PubMed Central

    Müller, Julia; Heuer, Heike

    2014-01-01

    For a comprehensive description of the tissue-specific thyroidal state under normal as well as under pathophysiological conditions it is of utmost importance to include thyroid hormone (TH) transporters in the analysis as well. The current knowledge of the cell-specific repertoire of TH transporters, however, is still rather limited, although several TH transporting proteins have been identified. Here, we describe the temporal and spatial distribution pattern of the most prominent TH transporters in the postnatal mouse brain. For that purpose, we performed radioactive in situ hybridization studies in order to analyze the cellular mRNA expression pattern of the monocarboxylate transporters Mct8 and Mct10, the L-type amino acid transporters Lat1 and Lat2 as well as the organic anion transporting peptide Oatp1c1 at different postnatal time points. Highest TH transporter expression levels in the CNS were observed at postnatal day 6 and 12, while hybridization signal intensities visibly declined after the second postnatal week. The only exception was Mct10 for which the strongest signals could be observed in white matter regions at postnatal day 21 indicating that this transporter is preferentially expressed in mature oligodendrocytes. Whereas Mct8 and Lat2 showed an overlapping neuronal mRNA expression pattern in the cerebral cortex, hippocampus, and in the hypothalamus, Oatp1c1 and Lat1 specific signals were most prominent in capillary endothelial cells throughout the CNS. In the choroid plexus, expression of three transporters (Mct8, Lat2, and Oatp1c1) could be detected, whereas in other brain areas (e.g., striatum, thalamus, and brain stem nuclei) only one of the transporter candidates appeared to be present. Overall, our study revealed a distinct mRNA distribution pattern for each of the TH transporter candidates. Further studies will reveal to which extent these transporters contribute to the cell-specific TH uptake and efflux in the mouse CNS. PMID:24994998

  10. Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

    SciTech Connect

    Al-Dosari, Mohammed; Zhang Guisheng; Knapp, Joseph E.; Liu Dexi . E-mail: dliu@pitt.edu

    2006-01-13

    Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), {alpha}-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken {beta} actin (ACT), nuclear factor {kappa} B (NF{kappa}B), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.

  11. Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

    PubMed

    Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

    2016-04-01

    Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.

  12. Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse

    PubMed Central

    Paul, Erin; Kielbasinski, Marissa; Sedivy, John M.; Murga-Zamalloa, Carlos; Khanna, Hemant; Klysik, Jan E.

    2009-01-01

    Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression. PMID:19937380

  13. Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

    PubMed Central

    Xie, Wen; Li, Fugang; Kudlow, Jeffrey E.; Wu, Chuanyue

    1998-01-01

    Integrin-linked kinase (ILK) is a newly identified serine/threonine protein kinase implicated in integrin signaling. To investigate the functions of ILK in vivo, we have analyzed the expression and regulation of ILK in the skin, in which proper control of cell-extracellular matrix interactions and cell proliferation is essential for its normal development and homeostasis. We report here that ILK is abundantly expressed throughout the extracellular matrix-rich dermis. ILK mRNA was also detected in the hair follicles and the basal cells of the interfollicular epidermis. However, ILK expression is lost in the suprabasal layers of keratinocytes that are undergoing terminal differentiation. PINCH, an ILK-binding protein, exhibited a similar expression pattern in the skin. Recent studies have indicated that erbB-2, a member of the epidermal growth factor receptor family, plays a pivotal role in epidermal growth, differentiation, and hair follicle morphogenesis. Using a transgenic mouse system in which an activated erbB-2 is overexpressed in the epidermis, we show that ILK expression is regulated by erbB-2. The in vivo expression and regulation patterns of ILK, together with its biochemical activities, suggest an important role of ILK in coordinating the integrin signaling pathways and the growth factor signaling pathways in the development of the skin and the pathogenesis of skin diseases. PMID:9708797

  14. Prolactin effects on the dietary regulation of mouse mammary tumor virus proviral DNA expression.

    PubMed Central

    Hamada, N; Engelman, R W; Tomita, Y; Chen, R F; Iwai, H; Good, R A; Day, N K

    1990-01-01

    Chronic energy-intake restriction inhibits mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice by greater than 90%. We have shown that associated with suppression of mammary tumorigenesis there is a reduction or inhibition of circulating prolactin, MMTV particles expressed, and MMTV mRNA transcription in mammary glands (and in most organs tested). To understand the concerted action of prolactin, energy-consumption level, and MMTV on inducing mammary tumors, experiments were designed to control prolactin and energy levels in order to evaluate their effects on MMTV mRNA expression. Mice on restricted diets were grafted with adenohypophyses, and mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo [g]quinoline. Adenohypophyseal grafting significantly increased prolactin in dietary (energy)-restricted mice, and this effect was associated with an increase in MMTV mRNA expression within the mammary gland; a linear correlation between prolactin levels and MMTV mRNA expression in the mammary gland was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of dopaminomimetic octahydrobenzo [g]quinoline to mice fed ad libitum delayed (by 8 weeks) and reduced (even as long as 25 weeks) mammary gland MMTV mRNA expression. These findings associate prolactin influences with MMTV mRNA production in mice and help explain the link between chronic energy-intake restriction and reduced MMTV gene expression. Images PMID:1975696

  15. Differential spatiotemporal expression of E- and P-cadherin during mouse tooth development.

    PubMed

    Palacios, J; Benito, N; Berraquero, R; Pizarro, A; Cano, A; Gamallo, C

    1995-08-01

    Changes in E- and P-cadherin (E- and P-CD) expression during embryonic mouse first molar development were analyzed by immunohistochemistry. During the induction and morphogenesis stages (bud, cap and early bell stages), E-CD was expressed in the cells of the invaginating epithelial tooth bud and in the cells of the outer enamel epithelium, stellate reticulum and stratum intermedium, suggesting a role for this molecule in the maintenance of enamel organ architecture. On the other hand, P-CD was strongly expressed in the inner enamel epithelium suggesting its participation in the processes of mesenchymal induction. during the cytodifferentiation stage (late bell stage), E-CD was expressed in polarizing preameloblasts, but cadherin expression was restricted to the basal and apical poles of differentiated secretory ameloblasts, where the zonula adherens type of cell-cell junctions is located. The present study demonstrates for the first time the spatiotemporal expression of cadherins during tooth development and suggests differential and specific roles for E-CD and P-CD during the morphogenesis and cytodifferentiation processes of this organ.

  16. Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

    PubMed Central

    2012-01-01

    Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis. PMID:22860215

  17. Aquaporin-4 expression contributes to decreases in brain water content during mouse postnatal development.

    PubMed

    Li, Xiumiao; Gao, Junying; Ding, Jiong; Hu, Gang; Xiao, Ming

    2013-05-01

    The water channel protein aquaporin-4 (AQP4) is implicated to facilitate water efflux from the brain parenchyma into the blood and CSF, playing a critical role in maintaining brain water homeostasis. Nevertheless, its contribution to decreases in brain water content during postnatal development remains unknown. A quantitative Western blot analysis was performed to investigate developmental expression of AQP4 in the whole mouse brain and showed that AQP4 expression level in 1 week-old brain was only 21.3% of that in the adult brain, but significantly increased to 67.4% of the adult level by 2 weeks after birth. Statistical analysis demonstrated that increased AQP4 expression partially relates to decreased brain water content in postnatal mice (r(2)=0.92 and P=0.002). Moreover, AQP4 null mice had greater brain water content than littermate controls from 2 weeks up to adult age. Consistently, mature pattern of AQP4 localization at the brain-blood and brain-CSF interfaces were completed at approximately at 2 weeks after birth. In addition, AQP4 expression in the brain stem and hypothalamus was earlier than that in the cerebral cortex and cerebellum, suggesting a brain regional variation in developmental expression of AQP4. These results characterize the developmental feature of AQP4 expression in the postnatal brain and provide direct evidence for a role of AQP4 in postnatal brain water uptake.

  18. Antisense oligonucleotides delivered to the amniotic cavity in utero modulate gene expression in the postnatal mouse

    PubMed Central

    Depreux, Frederic F.; Wang, Lingyan; Jiang, Han; Jodelka, Francine M.; Rosencrans, Robert F.; Rigo, Frank; Lentz, Jennifer J.; Brigande, John V.; Hastings, Michelle L.

    2016-01-01

    Congenital diseases account for a large portion of pediatric illness. Prenatal screening and diagnosis permit early detection of many genetic diseases. Fetal therapeutic strategies to manage disease processes in utero represent a powerful new approach for clinical care. A safe and effective fetal pharmacotherapy designed to modulate gene expression ideally would avoid direct mechanical engagement of the fetus and present an external reservoir of drug. The amniotic cavity surrounding the fetus could serve as an ideal drug reservoir. Antisense oligonucleotides (ASOs) are an established tool for the therapeutic modulation of gene expression. We hypothesize that ASOs administered to the amniotic cavity will gain entry to the fetus and modulate gene expression. Here, we show that an ASO targeting MALAT1 RNA, delivered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13-13.5, reduces target RNA expression for up to 4 weeks after birth. A similarly delivered ASO targeting a causal splice site mutation for Usher syndrome corrects gene expression in the inner ear, a therapeutically relevant target tissue. We conclude that intra-amniotic delivery of ASOs is well tolerated and produces a sustained effect on postnatal gene expression. Transuterine delivery of ASOs is an innovative platform for developing fetal therapeutics to efficaciously treat congenital disease. PMID:27683224

  19. An analysis of the expression of cyclophilin C reveals tissue restriction and an intriguing pattern in the mouse kidney.

    PubMed Central

    Friedman, J.; Weissman, I.; Friedman, J.; Alpert, S.

    1994-01-01

    Cyclophilin C (cyp C) is a cyclosporin A (CsA) binding protein originally isolated from a mouse bone marrow stromal cell line. We have compared the expression patterns of the mammalian cyclophilins A, B, and C in mouse tissues using in situ hybridization. These studies reveal that cyp C is expressed in a restricted subset of tissues including mouse ovary, testis, bone marrow, and kidney. Within the kidney, cyp C is highly expressed in a narrow zone in the outer medulla. Using monoclonal antibodies reactive against cyp C, we find that the kidney cells expressing cyp C correspond to the S3 segment of the nephron. The S3 segment has been shown to sustain histopathological damage from high dosages of CsA, raising the possibility that cyp C may be involved in mediating this damage. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8203464

  20. Developmental Gene Expression Profiling along the Tonotopic Axis of the Mouse Cochlea

    PubMed Central

    Son, Eun Jin; Wu, Ling; Yoon, Heejei; Kim, Sunhee; Choi, Jae Young; Bok, Jinwoong

    2012-01-01

    The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. Tnc (tenacin C) and Nov (nephroblastoma overexpressed gene) are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, Fst (Follistatin), an antagonist of TGF-β/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of Fst is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy. PMID:22808246

  1. Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut.

    PubMed

    Zhang, S; Racaniello, V R

    1997-07-01

    Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.

  2. Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

    PubMed

    Li, Ya-Jing; ZhuGe, Fu-Yan; Zeng, Chang-Chun; He, Jin-Yang; Tan, Ning; Liang, Juan

    2017-04-01

    A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

  3. Apoptosis and gene expression in the developing mouse brain of fusarenon-X-treated pregnant mice.

    PubMed

    Sutjarit, Samak; Nakayama, Shota M M; Ikenaka, Yoshinori; Ishizuka, Mayumi; Banlunara, Wijit; Rerkamnuaychoke, Worawut; Kumagai, Susumu; Poapolathep, Amnart

    2014-08-17

    Fusarenon-X (FX), a type B trichothecene mycotoxin, is mainly produced by Fusarium crookwellense, which occurs naturally in agricultural commodities, such as wheat and barley. FX has been shown to exert a variety of toxic effects on multiple targets in vitro. However, the embryonic toxicity of FX in vivo remains unclear. In the present study, we investigated FX-induced apoptosis and the relationship between the genetic regulatory mechanisms and FX-induced apoptosis in the developing mouse brain of FX-treated pregnant mice. Pregnant mice were orally administered FX (3.5 mg/kg b.w.) and were assessed at 0, 12, 24 and 48 h after treatment (HAT). Apoptosis in the fetal brain was determined using hematoxylin and eosin staining, the TUNEL method, immunohistochemistry for PCNA and electron microscopy. Gene expressions were evaluated using microarray and real time-reverse transcription polymerase chain reaction (qRT-PCR). Histopathological changes showed that the number of apoptotic cells in the telencephalon of the mouse fetus peaked at 12 HAT and decreased at 24 and 48 HAT. FX induced the up-regulation of Bax, Trp53 and Casp9 and down-regulated Bcl2 but the expression levels of Fas and Casp8 mRNA remained unchanged. These data suggested that FX induces apoptosis in the developing mouse brain in FX-treated dams. Moreover, the genetic regulatory mechanisms of FX-induced apoptosis are regulated by Bax, Bcl2, Trp53 and Casp9 or can be defined via an intrinsic apoptotic pathway.

  4. High-Throughput Analysis of Dynamic Gene Expression Associated with Sleep Deprivation and Recovery Sleep in the Mouse Brain

    DTIC Science & Technology

    2006-12-01

    Gene Expression Associated with Sleep Deprivation and Recovery Sleep in the Mouse Brain PRINCIPAL INVESTIGATOR: Ed Lein, Ph.D...CONTRACTING ORGANIZATION: Allen Institute for Brain Science Seattle, WA 98103 REPORT DATE...Recovery Sleep in the Mouse Brain 5b. GRANT NUMBER W81XWH-06-1-0131 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Ed Lein, Ph.D

  5. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    SciTech Connect

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R.

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  6. An acid phosphatase locus expressed in mouse kidney (Apk) and its genetic location on chromosome 10.

    PubMed

    Womack, J E; Auerbach, S B

    1978-04-01

    A genetic locus controlling the electrophoretic mobility of an acid phosphatase in mouse kidney is described. This locus, called acid phosphatase-kidney (Apk), is not expressed in erythrocytes, liver, spleen, heart, lung, brain, skeletal muscle, stomach, or testes. The product of Apk hydrolyzes the substrate naphthol AS-MX phosphoric acid but is not active on alpha-naphthylphosphate or 4-methylumbelliferylphosphate. It is not inactivated by 50 C for 1 hr, nor is its electrophoretic mobility altered by incubation with neuraminidase. The locus is invariant among 31 inbred strains (Apka), with a variant allele (Apkm) observed only in Mus musculus molossinus. Codominant expression was observed in F1 hybrids of M. m. molossinus and inbred strains. Apk was mapped on Chr 10, near the neurological mutant waltzer (v).

  7. Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

    PubMed Central

    Du, Hongqing; Cline, Melissa S.; Osborne, Robert J.; Tuttle, Daniel L.; Clark, Tyson A.; Donohue, John Paul; Hall, Megan P.; Shiue, Lily; Swanson, Maurice S.; Thornton, Charles A.; Ares, Manuel

    2009-01-01

    Myotonic dystrophy (DM1) is associated with expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. Strong correlation in splicing changes for ~100 new Mbnl1-regulated exons indicates loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA level changes can be attributed to loss of Mbnl1, indicating CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix (ECM) proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function, disrupting splicing, and loss of another function that disrupts ECM mRNA regulation, possibly mediated by MBNL2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies. PMID:20098426

  8. Nestin Expression in the Adult Mouse Retina with Pharmaceutically Induced Retinal Degeneration

    PubMed Central

    2017-01-01

    The present study investigated the temporal pattern and cellular localization of nestin in the adult mouse retina with pharmaceutically induced retinal degeneration using N-methyl-N-nitrosourea (MNU). After a single intraperitoneal injection of MNU in 8-week-old C57BL/6 mice, the animals were sacrificed at 1, 3, 5, 7, and 21 days (n = 6, in each stage). The eyes were examined by means of immunohistochemical tests using nestin, ionized calcium-binding adaptor molecule (Iba-1), CD11b, F4/80, and glial fibrillary acidic protein (GFAP). Western blot analysis and manual cell counting were performed for quantification. Nestin expression was increased after MNU administration. Nestin+/Iba-1+ cells were migrated into outer nuclear layer (ONL) and peaked at day 3 post injection (PI). Nestin+/CD11b+ cells were also mainly identified in ONL at day 3 PI and peaked at day 5. Nestin+/F4/80+ cells were shown in the subretinal space and peaked at day 3 PI. Nestin+/GFAP+ cells were distinctly increased at day 1 PI and peaked at day 5 PI. The up-regulation of nestin expression after MNU administration in adult mouse retinal microglia, and monocyte/macrophage suggests that when retinal degeneration progresses, these cells may revert to a more developmentally immature state. Müller cells also showed reactive gliosis and differentiational changes. PMID:28049248

  9. Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.

    PubMed

    Huss, David; Choi, Harry M T; Readhead, Carol; Fraser, Scott E; Pierce, Niles A; Lansford, Rusty

    2015-03-02

    Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.

  10. Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

    PubMed Central

    Barraud-Lange, Virginie; Naud-Barriant, Nathalie; Saffar, Line; Gattegno, Liliane; Ducot, Beatrice; Drillet, Anne-Sophie; Bomsel, Morgane; Wolf, Jean-Philippe; Ziyyat, Ahmed

    2007-01-01

    Background Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. Results Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. Conclusion These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported. PMID:17850654

  11. Cardiac fibrosis in mouse expressing DsRed tetramers involves chronic autophagy and proteasome degradation insufficiency

    PubMed Central

    Chen, Tsung-Hsien; Chen, Mei-Ru; Chen, Tzu-Yin; Wu, Tzu-Chin; Liu, Shan-Wen; Hsu, Ching-Han; Liou, Gan-Guang; Kao, Yu-Ying; Dong, Guo-Chung; Chu, Pao-Hsien; Liao, Jiunn-Wang; Lin, Kurt Ming-Chao

    2016-01-01

    Proteinopathy in the heart which often manifests excessive misfolded/aggregated proteins in cardiac myocytes can result in severe fibrosis and heart failure. Here we developed a mouse model, which transgenically express tetrameric DsRed, a red fluorescent protein (RFP), in an attempt to mimic the pathological mechanisms ofcardiac fibrosis. Whilst DsRed is expressed and forms aggregation in most mouse organs, certain pathological defects are specifically recapitulated in cardiac muscle cells including mitochondria damages, aggresome-like residual bodies, excessive ubiquitinated proteins, and the induction of autophagy. The proteinopathy and cellular injuries caused by DsRed aggregates may be due to impaired or overburdened ubiquitin-proteasome system and autophagy-lysosome systems. We further identified that DsRed can be ubiquitinated and associated with MuRF1, a muscle-specific E3 ligase. Concomitantly, an activation of NF-κB signaling and a strong TIMP1 induction were noted, suggesting that RFP-induced fibrosis was augmented by a skewed balance between TIMP1 and MMPs. Taken together, our study highlights the molecular consequences of uncontrolled protein aggregation leading to congestive heart failure, and provides novel insights into fibrosis formation that can be exploited for improved therapy. PMID:27494843

  12. Structure and expression of mouse mitochondrial voltage dependent anion channel genes

    SciTech Connect

    Craigen, W.J.; Lovell, R.S.; Sampson, M.J.

    1994-09-01

    Voltage dependent anion channels (VDACs) are small abundant proteins of the outer mitochondrial membrane that interact with the adenine nucleotide translocater and bind glycerol kinase and hexokinase. Kinase binding is developmentally regulated, tissue specific, and increased in various tumor cell lines. VDACs are also components of the peripheral benzodiazepine receptor and GABA{sub A} receptor. Two human VDAC cDNAs have previously been reported, and expression of these isoforms appears ubiquitous. Genomic Southern analysis suggests the presence of other as yet uncharacterised VDAC genes. To study VDAC function in a mammal more amenable to experimental manipulation, we have isolated three mouse VDAC genes by cDNA cloning from a mouse brain cDNA library. DNA sequencing of the cDNAs shows that they share 65-75% amino acid identity. Northern analysis indicates that MVDAC1 is expressed most highly in kidney, heart, and brain. Using an MVDAC3 3{prime} untranslated exon as a probe, three distinct transcripts can be detected. The gene structure for MVDAC3 and MVDAC2 has been completed and suggests that the VDAC isoforms did not arise by gene duplication and divergence. The intron/exon boundaries are not conserved between MVDAC1 and MVDAC3, and MVDAC2 appears to be encoded by a single intronless gene.

  13. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

  14. Gene expression profiles in liver of mouse after chronic exposure to drinking water.

    PubMed

    Wu, Bing; Zhang, Yan; Zhao, Dayong; Zhang, Xuxiang; Kong, Zhiming; Cheng, Shupei

    2009-10-01

    cDNA micorarray approach was applied to hepatic transcriptional profile analysis in male mouse (Mus musculus, ICR) to assess the potential health effects of drinking water in Nanjing, China. Mice were treated with continuous exposure to drinking water for 90 days. Hepatic gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 arrays, and pathway analysis was carried out by Molecule Annotation System 2.0 and KEGG pathway database. A total of 836 genes were found to be significantly altered (1.5-fold, P < or = 0.05), including 294 up-regulated genes and 542 down-regulated genes. According to biological pathway analysis, drinking water exposure resulted in aberration of gene expression and biological pathways linked to xenobiotic metabolism, signal transduction, cell cycle and oxidative stress response. Further, deregulation of several genes associated with carcinogenesis or tumor progression including Ccnd1, Egfr, Map2k3, Mcm2, Orc2l and Smad2 was observed. Although transcription changes in identified genes are unlikely to be used as a sole indicator of adverse health effects, the results of this study could enhance our understanding of early toxic effects of drinking water exposure and support future studies on drinking water safety.

  15. CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

    PubMed Central

    Zhu, Quan; Gregg, Keqin; Lozano, Mary; Liu, Jinqi; Dudley, Jaquelin P.

    2000-01-01

    Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland. PMID:10864645

  16. Melatonin-related genes expressed in the mouse uterus during early gestation promote embryo implantation.

    PubMed

    He, Changjiu; Wang, Jing; Li, Yu; Zhu, Kuanfeng; Xu, Zhiyuan; Song, Yile; Song, Yukun; Liu, Guoshi

    2015-04-01

    Melatonin, a superior antioxidant, is an important molecule which regulates female reproduction due to its receptor-mediated and receptor-independent antioxidant actions. In this study, we investigated the effect of melatonin on early gestation in a mouse model. During early gestation, the expression of the melatonin's rate-limiting enzyme, AANAT, gradually increased - in the uterus while the MT2 melatonin receptor was only expressed at day 2 of gestation and no MT1 was detected. Based on these findings, we conducted a melatonin injection experiment which demonstrated that 15 mg/kg melatonin significantly improved the number of implantation sites and the litter size. Also, the blastocyst and uterus were collected to identify the local action of melatonin. In the melatonin-treated mice, the endometrium was thicker than in the control mice; melatonin also caused an increase in density of uterine glands, and the uterine gland index (UGI) was significantly elevated over that of the control. Serum steroid hormone measurements revealed that at day 6 of gestation (postimplantation), melatonin significantly downregulated the E2 level, with no obvious effects on progesterone. Gene expression assay revealed that melatonin significantly upregulated expression of HB-EGF, a crucial gene involved in implantation as well as its receptor ErbB1 in the blastocyst. In addition, PRA, an important gene which influences the decidual response and luminal cell differentiation, p53, which regulates uterine through leukaemia inhibitory factor (LIF), were both increased after melatonin treatment. These data suggest that melatonin and its MT2 receptor influence early gestation. Exogenous melatonin treatment can improve mouse embryo implantation and litter size, which may have important applications in human reproductive health and animal husbandry.

  17. Growth Arrest Specific 1 (GAS1) Is Abundantly Expressed in the Adult Mouse Central Nervous System

    PubMed Central

    Zarco, Natanael; Bautista, Elizabeth; Cuéllar, Manola; Vergara, Paula; Flores-Rodriguez, Paola; Aguilar-Roblero, Raúl

    2013-01-01

    Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS. PMID:23813868

  18. Novel Variants of Oct-3/4 Gene Expressed in Mouse Somatic Cells*S⃞

    PubMed Central

    Mizuno, Nobuhiko; Kosaka, Mitsuko

    2008-01-01

    It has been suggested that Oct-3/4 may regulate self-renewal in somatic stem cells, as it does in embryonic stem cells. However, recent reports raise the possibility that detection of human Oct-3/4 expression by RT-PCR is prone to artifacts generated by pseudogene transcripts and argue against a role for Oct-3/4 in somatic cells. In this study, we clarified Oct-3/4 expression in mouse somatic tissues using designed PCR primers, which can exclude amplification of its pseudogenes. We found that novel alternative transcripts are indeed expressed in somatic tissues, rather than the normal length transcripts in germline and ES cells. The alternative transcripts indicate the expression of two kinds of truncated proteins. Furthermore, we determined novel promoter regions that are sufficient for the expression of Oct-3/4 transcript variants in somatic cells. These findings provide new insights into the postnatal role of Oct-3/4 in somatic tissues. PMID:18765667

  19. Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse.

    PubMed

    Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

    2017-03-05

    Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS.

  20. Gene expression analysis of embryonic photoreceptor precursor cells using BAC-Crx-EGFP transgenic mouse.

    PubMed

    Muranishi, Yuki; Sato, Shigeru; Inoue, Tatsuya; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

    2010-02-12

    Crx is a transcription factor which is predominantly expressed in developing and mature photoreceptor cells in the retina, and plays a crucial role in the terminal differentiation of both rods and cones. Crx is one of the earliest-expressed genes specifically in photoreceptor precursors, allowing us to trace photoreceptor precursor cells from embryonic stages to adult stage by visualizing Crx-expressing cells. In the current study, we generated a transgenic mouse line which expresses enhanced green fluorescence protein (EGFP) in the retina driven by the Crx promoter using bacterial artificial chromosome (BAC) transgenesis. EGFP-positive cells were observed in the presumptive photoreceptor layer in the retina at embryonic day 15.5 (E15.5), and continued to be expressed in developing and mature photoreceptor cells up to adult stage. We sorted EGFP-positive photoreceptor precursors at E17.5 using fluorescence-activated cell sorter (FACS), and subsequently performed microarray analysis of the FACS-sorted cells. We observed various photoreceptor genes, especially cone genes, are enriched in the EGFP-positive cells, indicating that embryonic cone photoreceptor precursors are enriched. In addition, we found that most of the EGFP-positive cells were post-mitotic cells. Thus, the transgenic line we established can serve as a useful tool to study both developing and mature photoreceptor cells, including embryonic cone precursors whose analysis has been difficult.

  1. Radiation effects on cellularity, proliferation and EGFR expression in mouse bladder urothelium.

    PubMed

    Jaal, Jana; Dörr, Wolfgang

    2010-04-01

    This study was designed to determine changes in cell numbers, proliferation (using Ki-67) and EGFR expression in mouse bladder urothelium during the early and late radiation response. Groups of mice were irradiated with a single dose of 20 Gy and assayed 0-360 days later. Urothelial cells were counted. After immunohistochemistry, the absolute and relative numbers of Ki-67(+) and EGFR(+) cells were analyzed. Radiation exposure resulted in a decrease in total urothelial cell numbers to 49% by day 31, with restoration of cellularity by day 180. In contrast, at day 360, an increase in total cell number (143%) was seen. Slightly increased Ki-67 expression was found at days 120 and 180 after treatment, followed by a pronounced elevation at days 240 and 360. Compared to controls, higher EGFR expression was detected up to day 360 after irradiation. A positive correlation was found between total urothelial cells numbers and Ki-67 as well as EGFR expression. Radiation exposure results in an increased urothelial expression of EGFR that precedes urothelial restoration, indicating a contribution of the EGF/EGFR system to urothelial proliferation and differentiation. Further studies are needed to evaluate the impact of EGFR inhibition on radiation effects in the urinary bladder.

  2. Developmental and cell-specific expression of thyroid hormone transporters in the mouse cochlea.

    PubMed

    Sharlin, David S; Visser, Theo J; Forrest, Douglas

    2011-12-01

    Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr(-/-) mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development.

  3. Fezf2 expression in layer 5 projection neurons of mature mouse motor cortex.

    PubMed

    Tantirigama, Malinda L S; Oswald, Manfred J; Clare, Alison J; Wicky, Hollie E; Day, Robert C; Hughes, Stephanie M; Empson, Ruth M

    2016-03-01

    The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.

  4. Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse

    PubMed Central

    Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

    2017-01-01

    Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS. PMID:28273875

  5. Chicken ovalbumin upstream promoter transcription factor (COUP-TF): expression during mouse embryogenesis.

    PubMed

    Pereira, F A; Qiu, Y; Tsai, M J; Tsai, S Y

    1995-06-01

    Members of the steroid/thyroid hormone receptor superfamily such as TR, RAR, RXR and VDR are known to play important roles in regulation of gene expression during development, differentiation and homeostasis. COUP-TFs are orphan members of this superfamily of nuclear receptors and have been shown to negatively regulate the ability of these nuclear receptors to transactivate target genes. Two different mechanisms are implicated in this repression. First, COUP-TFs bind to AGGTCA direct repeats and palindromes with various spacings, which include response elements for TR, RAR, RXR and VDR, allowing for direct competition of COUP-TFs for the response elements. Second, COUP-TFs can heterodimerize with RXRs, the essential cofactor for effective binding of VDR, TRs and RARs to their cognate response elements. The physiological significance of this negative effect of COUP-TF on the activity of these receptors has been analyzed. Detection of COUP-TF transcripts during mouse development reveal discrete spatial and temporal expression domains consistent with COUP-TFs being involved in regulation of gene expression during embryogenesis. Transcripts are localized within discrete regions of the central and peripheral nervous system including the inner ear. In addition, COUP-TFs are found in many tissues including testes, ovary, prostate, skin, kidney, lung, stomach, intestine, pancreas and salivary gland. Some of these expression domains colocalize with those of TR, RAR, and RXR. The simultaneous expression of these genes raise the possibility that COUP-TFs can act as negative regulatory factors during development and differentiation.

  6. Absence of MHC gene expression in lens and cloning of dbpB/YB-1, a DNA-binding protein expressed in mouse lens.

    PubMed

    Shaughnessy, M; Wistow, G

    1992-02-01

    The status of major histocompatibility complex (MHC) class I and II gene expression in the normal mouse lens was examined. No mRNA for either class I or II genes was detectable in mouse lens, while the expression of MHC genes in other tissues generally matched immunohistochemical data from human tissues. However it was observed that MHC class I mRNA is present in the mouse lens-derived cell line alpha TN4-1. From a new-born mouse lens cDNA library a clone was obtained for the murine homologue of the DNA-binding protein dbpB/YB-1, a protein originally identified in human lymphocytes and proposed to be a negative regulator of MHC class II gene expression. Northern blots detect dbpB/YB-1 mRNA in all mouse tissues and cells examined, including both mouse lens and alpha TN4-1 cells, suggesting that dbpB/YB-1 has a general and widespread role.

  7. Regulated expression of mouse mammary tumor proviral genes in cells of the B lineage

    PubMed Central

    1991-01-01

    We evaluated the expression of mouse mammary tumor proviral (MMTV) transcripts during B cell ontogeny and compared levels of RNA in B lymphocytes and B cell lines with levels in other cells of the hematopoietic lineage and in a mammary cell line. We demonstrate that MMTV transcripts are expressed as early as the pro-B cell stage in ontogeny and are expressed at basal constitutive levels throughout most of the B cell developmental pathway. The level of MMTV expression in B cells is similar to constitutive levels in mammary tissues and two to three orders of magnitude greater than in activated T cells. Levels of MMTV transcripts in B cells are not solely due to positional effects. Transient transfection assays showed that MMTV upregulation resulted from transcriptional activation of the viral LTR, indicating that there are specific and inducible transcription factors that regulate MMTV expression in B cells. MMTV transcripts could not be upregulated in pre- B cell lines but could be induced in some mature B cell lines. There was a correlation between the ability to stimulate B cells to secrete antibody and the ability to induce upregulated MMTV expression. Evidence is presented that suggests that the principal transcription factors involved in MMTV expression do not include the B cell factors OTF-2 or NF-kappa B, but rather are likely to be novel factors that are induced during differentiation to antibody secretion. A hypothesis for why mammary tumor viruses are well adapted for expression in cells of the B lineage is proposed, and the implications of this for the documented influence of MMTV gene products on the T cell repertoire are discussed. PMID:1660524

  8. Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

    PubMed

    Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

    2012-11-01

    Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

  9. Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development.

    PubMed Central

    Soifer, S. J.; Peters, K. G.; O'Keefe, J.; Coughlin, S. R.

    1994-01-01

    The protease thrombin is a potent agonist for platelet aggregation, mesenchymal cell proliferation, and endothelial production of growth factors and adhesion molecules. Thrombin also modulates neurite outgrowth in neuronal cultures. These apparently disparate responses to thrombin appear to be largely mediated by the recently cloned thrombin receptor. In the adult, thrombin is generated from its zymogen prothrombin at sites of vascular injury when circulating coagulation factors meet extravascular tissue factor. In this context thrombin's varied actions may mediate responses to wounding. Whether thrombin's actions on cells may also play a role in development is unknown. We examined the expression of thrombin receptor, prothrombin, and tissue factor by in situ hybridization in mouse development. Thrombin receptor mRNA was expressed widely in mesenchymal cell populations during early organogenesis (E9.5) and was particularly abundant in developing heart and blood vessels. Robust receptor expression was also noted in the germinal epithelium of the hindbrain. Thrombin receptor expression became more restricted with time and by the fetal growth stage (E16.5) was most readily detected in certain neurons, endocardial and endothelial cells, and within lung and liver. In contrast to the thrombin receptor, prothrombin mRNA was limited to the embryonic liver and was not detected until E12.5, well after the onset of receptor expression. mRNA for tissue factor, one important trigger for thrombin generation in the adult, was detected in embryonic epithelia from E9.5-12.5. In several instances, tissue factor-expressing epithelia were surrounded by thrombin receptor-expressing mesenchyme. These data suggest a possible role for the thrombin receptor in development. The finding of robust thrombin receptor expression before prothrombin mRNA was detected raises the question of whether other proteases or peptide ligands can activate the thrombin receptor. Images Figure 1 Figure 2

  10. Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis

    PubMed Central

    Garcia-Fabiani, Maria B.; Montanaro, Mauro A.; Lacunza, Ezequiel; Cattaneo, Elizabeth R.; Coleman, Rosalind A.; Pellon-Maison, Magali; Gonzalez-Baro, Maria R.

    2015-01-01

    Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on–off expression pattern responds predominantly to epigenetic modifications. PMID:26268560

  11. Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans.

    PubMed Central

    Srivastava, Meera; Eidelman, Ofer; Leighton, Ximena; Glasman, Mirta; Goping, Gertrude; Pollard, Harvey B.

    2002-01-01

    BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control

  12. Quorum Sensing Inhibitors from the Sea Discovered Using Bacterial N-acyl-homoserine Lactone-Based Biosensors.

    PubMed

    Saurav, Kumar; Costantino, Valeria; Venturi, Vittorio; Steindler, Laura

    2017-02-23

    Marine natural products with antibiotic activity have been a rich source of drug discovery; however, the emergence of antibiotic-resistant bacterial strains has turned attention towards the discovery of alternative innovative strategies to combat pathogens. In many pathogenic bacteria, the expression of virulence factors is under the regulation of quorum sensing (QS). QS inhibitors (QSIs) present a promising alternative or potential synergistic treatment since they disrupt the signaling pathway used for intra- and interspecies coordination of expression of virulence factors. This review covers the set of molecules showing QSI activity that were isolated from marine organisms, including plants (algae), animals (sponges, cnidarians, and bryozoans), and microorganisms (bacteria, fungi, and cyanobacteria). The compounds found and the methods used for their isolation are the emphasis of this review.

  13. Quorum Sensing Inhibitors from the Sea Discovered Using Bacterial N-acyl-homoserine Lactone-Based Biosensors

    PubMed Central

    Saurav, Kumar; Costantino, Valeria; Venturi, Vittorio; Steindler, Laura

    2017-01-01

    Marine natural products with antibiotic activity have been a rich source of drug discovery; however, the emergence of antibiotic-resistant bacterial strains has turned attention towards the discovery of alternative innovative strategies to combat pathogens. In many pathogenic bacteria, the expression of virulence factors is under the regulation of quorum sensing (QS). QS inhibitors (QSIs) present a promising alternative or potential synergistic treatment since they disrupt the signaling pathway used for intra- and interspecies coordination of expression of virulence factors. This review covers the set of molecules showing QSI activity that were isolated from marine organisms, including plants (algae), animals (sponges, cnidarians, and bryozoans), and microorganisms (bacteria, fungi, and cyanobacteria). The compounds found and the methods used for their isolation are the emphasis of this review. PMID:28241461

  14. Neuronal Expression of CB2 Cannabinoid Receptor mRNAs in the Mouse Hippocampus

    PubMed Central

    Li, Yong; Kim, Jimok

    2015-01-01

    In the brain, CB1 cannabinoid receptors primarily mediate the effects of cannabinoids, but CB2 cannabinoid receptors (CB2Rs) have recently been discovered in the nervous system and also implicated in neuromodulatory roles. To understand the mechanisms of CB2R functions in the brain, it is essential to localize CB2Rs, but the types of cells expressing CB2Rs have been controversial. Unequivocal localization of CB2Rs in the brain has been impeded in part by the low expression levels of CB2Rs and poor specificity of detection methods. Here, we used an ultrasensitive and specific in situ hybridization method called the RNAscope to determine the spatial pattern of CB2R mRNA expression in the mouse hippocampus. CB2R mRNAs were mostly expressed in a subset of excitatory and inhibitory neurons in the CA1, CA3 and dentate gyrus areas, but rarely in microglia. CB2R knock-out mice were used as a negative control. Using the quantitative real-time polymerase chain reaction, we also found that the temporal pattern of CB2R mRNA expression was stable during postnatal development. Consistent with previous reports, the immunological detection of CB2Rs was not reliable, implying extremely low levels of the protein expression and/or insufficient specificity of the current anti-CB2R antibodies. Our findings of the expression patterns of CB2R mRNAs may help determine the cell types involved in, and hence the mechanisms of, the CB2R-mediated neuromodulation. PMID:26515747

  15. Dynamic expression of Lgr6 in the developing and mature mouse cochlea

    PubMed Central

    Zhang, Yanping; Chen, Yan; Ni, Wenli; Guo, Luo; Lu, Xiaoling; Liu, Liman; Li, Wen; Sun, Shan; Wang, Lei; Li, Huawei

    2015-01-01

    The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5). From E18.5 to postnatal day 3 (P3), the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs). From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs). At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea. PMID:26029045

  16. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

  17. Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney.

    PubMed

    Graca, J A Z; Schepelmann, M; Brennan, S C; Reens, J; Chang, W; Yan, P; Toka, H; Riccardi, D; Price, S A

    2016-03-15

    The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca(2+)]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

  18. Comparative analysis of expression of histone H2a genes in mouse

    PubMed Central

    Nishida, Hiromi; Suzuki, Takahiro; Ookawa, Hiroki; Tomaru, Yasuhiro; Hayashizaki, Yoshihide

    2005-01-01

    Background At least 18 replication-dependent histone H2a genes are distributed in 3 Hist gene clusters on different chromosomes of the mouse genome. In this analysis we designed specific PCR primers for each histone H2a transcript and studied the expression levels and patterns using quantitative RT-PCR (qRT-PCR). In addition, we compared histone H3 K9 acetylation levels in the promoter regions of H2a genes by ChIP (chromatin immunoprecipitation) – quantitative PCR (qPCR) analysis. Results RT-PCR analysis indicated that all 20 histone H2a genes assessed in this study are expressed. The replication-dependent histone H2a genes have different expression levels but similar expression patterns. Among the 20 histone H2a genes, the expression-level of H2afz, a replication-independent gene, was highest, and that of Hist1h2aa, a replication-dependent gene, was lowest. Among 18 replication-dependent H2a genes, the expression level of Hist3h2a was highest. The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in promoter regions of both H2afz and Hist3h2a are clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa is similar to that in the γ-satellite region. Conclusion These results strongly suggest that histone H3 K9 acetylation plays a role in the expression of histone genes. PMID:16098230

  19. Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

    PubMed Central

    Liu, Xiaoqin; Zhang, Zhijing; Ribelayga, Christophe P.

    2012-01-01

    Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue. PMID:23189207

  20. Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.

    PubMed

    Feng, Weiguo; Leach, Sonia M; Tipney, Hannah; Phang, Tzulip; Geraci, Mark; Spritz, Richard A; Hunter, Lawrence E; Williams, Trevor

    2009-12-16

    Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions - the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5-E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional

  1. Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences

    PubMed Central

    Feng, Weiguo; Leach, Sonia M.; Tipney, Hannah; Phang, Tzulip; Geraci, Mark; Spritz, Richard A.; Hunter, Lawrence E.; Williams, Trevor

    2009-01-01

    Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions – the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5–E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional

  2. Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo.

    PubMed

    Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan; Surh, Young-Joon

    2013-09-06

    Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

  3. Age affects gene expression in mouse spermatogonial stem/progenitor cells.

    PubMed

    Kokkinaki, Maria; Lee, Tin-Lap; He, Zuping; Jiang, Jiji; Golestaneh, Nady; Hofmann, Marie-Claude; Chan, Wai-Yee; Dym, Martin

    2010-06-01

    Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties.

  4. Expression of fatty acid binding proteins is altered in aged mouse brain.

    PubMed

    Pu, L; Igbavboa, U; Wood, W G; Roths, J B; Kier, A B; Spener, F; Schroeder, F

    1999-08-01

    Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.

  5. Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

    SciTech Connect

    Fu, Tzu-Yen; Chang, Chia-Che; Lin, Chun-Ting; Lai, Cong-Hao; Peng, Shao-Yu; Ko, Yi-Ju; Tang, Pin-Chi

    2011-02-15

    Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

  6. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    PubMed

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  7. Detection of differentially expressed genes in the early developmental stage of the mouse mandible.

    PubMed

    Yamaza, H; Matsuo, K; Kiyoshima, T; Shigemura, N; Kobayashi, I; Wada, H; Akamime, A; Sakai, H

    2001-06-01

    We previously examined the development of the mouse mandible, and demonstrated that odontogenesis occurs between embryonic day 10.5 (E10.5) and E12. Based on the histological findings, we performed cDNA subtraction between the E10.5 and E12 mandibles to detect any differentially expressed genes which might be involved in the initiation of odontogenesis. By sequencing, homology search and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), we thus found Pgk-1, Ccte, Hsp86, Nucleolin, Hsc73, Frg1, N-ras, Set alpha and Hsj2 from the E10.5 mandible, and E25, ATPase6, Mum2, Thymosin beta4 and L21 from the E12 mandible to be differentially expressed genes. These genes are functionally related to protein transport, signal transduction, transcription, translation and molecular chaperon activity. In situ hybridization analyses of Set alpha and E25 showed that Set alpha was detected in the tooth germ at E12 and E14.5, thus indicating a close relationship of this gene to odontogenesis. Meanwhile, the in situ signal of E25 was found in the muscular layer of the tongue, thus suggesting E25 to be related to the differentiation of muscular tissue. In conclusion, we found 15 differentially expressed genes in the course of the early developmental stage of the mouse mandible using a combination of the cDNA subtraction and semi-quantitative RT-PCR methods, while in addition, two genes were demonstrated to be related to the initiation and the development of both tooth germ and the tongue according to the in situ hybridization technique.

  8. Expression and regulation of lanosterol 14alpha-demethylase in mouse embryo and uterus during the peri-implantation period.

    PubMed

    Song, Xiaoming; Tai, Ping; Yan, Jun; Xu, Baoshan; Chen, Xiufen; Ouyang, Hong; Zhang, Meijia; Xia, Guoliang

    2008-01-01

    Lanosterol 14alpha-demethylase (LDM) is expressed ubiquitously in all mammals and is important in cholesterol biosynthesis. However, whether LDM expression is involved in the interaction between uterus and embryo during implantation remains unknown. In the present study, the expression of LDM was investigated in mouse embryo and uterus during the peri-implantation period using confocal microscopy, immunohistochemistry and western blot methods. Further, regulation of LDM expression was investigated in pseudopregnancy, delayed implantation, artificial decidualisation and ovariectomisation using 17beta-oestradiol and progesterone treatment mouse models. The results showed that LDM was selectively expressed in preimplantation embryos and the uterine subluminal stroma surrounding the implanting blastocyst on Day 5 of pregnancy. No corresponding signal was detected in the uterus on Day 5 of pseudopregnancy. Most notably, once delayed implantation was terminated by oestrogen treatment and the embryo implanted, a high level of LDM expression was induced in the subluminal stroma surrounding the implanting blastocyst, whereas no corresponding signal was detected in the delayed implantation uterus. A high level of LDM expression was observed in the uterus decidua on Days 6-8 of pregnancy. Furthermore, LDM expression was induced in the uterine stroma under artificial decidualisation. Oestrogen, but not progesterone, treatment induced a high level of LDM expression in the uterus of ovariectomised mice. These results indicate that LDM is closely related to mouse embryo implantation and can be upregulated by oestrogen.

  9. Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus

    PubMed Central

    Del Turco, Domenico; Paul, Mandy H.; Schlaudraff, Jessica; Hick, Meike; Endres, Kristina; Müller, Ulrike C.; Deller, Thomas

    2016-01-01

    The physiological role of amyloid precursor protein (APP) has been extensively investigated in the rodent hippocampus. Evidence suggests that APP plays a role in synaptic plasticity, dendritic and spine morphogenesis, neuroprotection and—at the behavioral level—hippocampus-dependent forms of learning and memory. Intriguingly, however, studies focusing on the role of APP in synaptic plasticity have reported diverging results and considerable differences in effect size between the dentate gyrus (DG) and area CA1 of the mouse hippocampus. We speculated that regional differences in APP expression could underlie these discrepancies and studied the expression of APP in both regions using immunostaining, in situ hybridization (ISH), and laser microdissection (LMD) in combination with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. In sum, our results show that APP is approximately 1.7-fold higher expressed in pyramidal cells of Ammon’s horn than in granule cells of the DG. This regional difference in APP expression may explain why loss-of-function approaches using APP-deficient mice revealed a role for APP in Hebbian plasticity in area CA1, whereas this could not be shown in the DG of the same APP mutants. PMID:27965537

  10. MDM2 expression during mouse embryogenesis and the requirement of p53.

    PubMed

    Léveillard, T; Gorry, P; Niederreither, K; Wasylyk, B

    1998-06-01

    We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

  11. Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells

    SciTech Connect

    Reyland, M.E.; Forgez, P.; Prack, M.M.; Williams, D.L. ); Gwynne, J.T. )

    1991-03-15

    The lipid transport protein, apolipoprotein E (apoE), is expressed in many peripheral tissues in vivo including the adrenal gland and testes. To investigate the role of apoE in adrenal cholesterol homeostasis, the authors have expressed a human apoE genomic clone in the Y1 mouse adrenocortical cell line. Y1 cells do not express endogenous apoE mRNA or protein. Expression of apoE in Y1 cells resulted in a dramatic decrease in basal steroidogenesis; secretion of fluorogenic steroid was reduced 7- to {gt}100-fold relative to Y1 parent cells. Addition of 5-cholesten-3{beta},25-idol failed to overcome the suppression of steroidogenesis in these cells. Cholesterol esterification under basal conditions, as measured by the production of cholesteryl ({sup 14}C)oleate, was similar in the Y1 parent and the apoE-transfected cell lines. Upon incubation with adrenocorticotropin or dibutyryl cAMP, production of cholesteryl ({sup 14}C)oleate decreased 5-fold in the Y1 parent cells but was unchanged in the apoE-transfected cell lines. These results suggest that apoE may be an important modulator of cholesterol utilization and steroidogenesis in adrenal cells.

  12. Onset of cell-specific gene expression in the developing mouse pancreas.

    PubMed Central

    Gittes, G K; Rutter, W J

    1992-01-01

    A central question in developmental biology has been the initiation of cell-specific gene expression and its temporal relationship to morphogenesis. We have coupled embryo microdissection with the exquisite sensitivity of the polymerase chain reaction to define the onset of cell-specific gene expression during pancreatic organogenesis. Using the precise assignment of gestational age by the number of somites in each embryo, we determined the onset of transcription of major genes of the endocrine and exocrine pancreas during mouse development to within 2-3 hr. Somatostatin mRNA was detected at the 10-somite stage throughout the foregut, consistent with the presence of somatostatin-producing cells throughout the adult gut. Mature mRNA for insulin and glucagon first appears surprisingly early, at the 20-somite stage in the wall of the embryonic foregut and is restricted to only the area of the duodenum from which the pancreas will arise 10-12 hr later. In contrast, exocrine gene transcription begins 24 hr after formation of the pancreatic diverticulum. Thus cell-specific gene expression in the endocrine pancreas begins in a "pre-morphogenetic phase." This early expression of insulin and glucagon could reflect the initiation of an endocrine cell lineage. Images PMID:1371010

  13. Structure and expression of the human and mouse T4 genes.

    PubMed Central

    Maddon, P J; Molineaux, S M; Maddon, D E; Zimmerman, K A; Godfrey, M; Alt, F W; Chess, L; Axel, R

    1987-01-01

    The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, we have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and a cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response. Images PMID:3501122

  14. Effects of atherogenic diet on hepatic gene expression across mouse strains

    PubMed Central

    Witmer, David; Burgess-Herbert, Sarah L.; Paigen, Beverly; Churchill, Gary A.

    2009-01-01

    Diets high in fat and cholesterol are associated with increased obesity and metabolic disease in mice and humans. To study the molecular basis of the metabolic response to dietary fat, 10 inbred strains of mice were fed atherogenic high-fat and control low-fat diets. Liver gene expression and whole animal phenotypes were measured and analyzed in both sexes. The effects of diet, strain, and sex on gene expression were determined irrespective of complex processes, such as feedback mechanisms, that could have mediated the genomic responses. Global gene expression analyses demonstrated that animals of the same strain and sex have similar transcriptional profiles on a low-fat diet, but strains may show considerable variability in response to high-fat diet. Functional profiling indicated that high-fat feeding induced genes in the immune response, indicating liver damage, and repressed cholesterol biosynthesis. The physiological significance of the transcriptional changes was confirmed by a correlation analysis of transcript levels with whole animal phenotypes. The results found here were used to confirm a previously identified quantitative trait locus on chromosome 17 identified in males fed a high-fat diet in two crosses, PERA × DBA/2 and PERA × I/Ln. The gene expression data and phenotype data have been made publicly available as an online tool for exploring the effects of atherogenic diet in inbred mouse strains (http://cgd-array.jax.org/DietStrainSurvey). PMID:19671657

  15. Expression of GLUT8 in mouse intestine: identification of alternative spliced variants.

    PubMed

    Romero, Amparo; Gomez, Olga; Terrado, Jose; Mesonero, Jose E

    2009-04-15

    GLUT8 is a facilitative glucose transporter composed of 10 exons coding for a 477 amino acids protein. It is mainly expressed in the testis, but it has also been studied in a number of tissues such as brain, adipose tissue, and liver. In this work, we have characterized the expression of GLUT8 in the small and large intestine under normal physiological conditions. Protein assay revealed low GLUT8 protein levels in the intestine compared to the testis, with higher levels in the colon than in the small intestine. Immunohistochemistry studies showed an intracellular localization of GLUT8 in enterocytes and colonocytes with a supranuclear distribution next to the apical membrane. GLUT8 immunoreactivity was also detected in the crypt cells. Interestingly, we have identified three additional transcriptional variants in mouse intestine (mGLUT-SP1, mGLUT8-SP2, and mGLUT8-SP3) produced by the deletion of one, two, and four exons, respectively, whereas only the entire mRNA was detected in the testis. Expression of these alternative variants did not have an effect on glucose consumption in 3T3-L1 cells. Although the specific function of GLUT8 in intestine remains unclear, the alternative splicing of GLUT8 could reflect a mechanism for the regulation of the gene expression in a tissue-specific manner by targeting GLUT8 mRNA for nonsense-mediated decay.

  16. Making the gradient: Thyroid hormone regulates cone opsin expression in the developing mouse retina

    PubMed Central

    Roberts, Melanie R.; Srinivas, Maya; Forrest, Douglas; Morreale de Escobar, Gabriella; Reh, Thomas A.

    2006-01-01

    Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal–ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone β2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRβ2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development. PMID:16606843

  17. Effect of chronic valproic Acid treatment on hepatic gene expression profile in wfs1 knockout mouse.

    PubMed

    Punapart, Marite; Eltermaa, Mall; Oflijan, Julia; Sütt, Silva; Must, Anne; Kõks, Sulev; Schalkwyk, Leonard C; Fernandes, Catherine; Vasar, Eero; Soomets, Ursel; Terasmaa, Anton

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

  18. Maternal stress affects postnatal growth and the pituitary expression of prolactin in mouse offspring.

    PubMed

    Gao, Pengfei; Ishige, Atsushi; Murakami, Yu; Nakata, Hideyuki; Oka, Jun-Ichiro; Munakata, Kaori; Yamamoto, Masahiro; Nishimura, Ko; Watanabe, Kenji

    2011-03-01

    Maternal stress exerts long-lasting psychiatric and somatic on offspring, which persist into adulthood. However, the effect of maternal stress on the postnatal growth of pups has not been widely reported. In this study, we found that maternal immobilization stress (IS) during lactation resulted in low body weight of male mouse offspring, which persisted after weaning. Despite free access to chow, IS induced maternal malnutrition and decreased the serum insulin-like growth factor-1 (IGF-1) levels in the mothers and in the pups. mRNA expression analysis of anterior pituitary hormones in the pups revealed that growth hormone (GH) and prolactin (PRL), but no other hormones, were decreased by IS. Expression of the pituitary transcription factor PIT1 and isoforms of PITX2, which are essential for the development and function of GH-producing somatotropes and PRL-producing lactotropes, was decreased, whereas that of PROP1, which is critical for the earlier stages of pituitary development, was unchanged. Immunohistochemistry also showed a decrease in pituitary PRL protein expression. These results suggest that stress in a postpartum mother has persistent effects on the body weight of the offspring. Reduced PRL expression in the offspring's pituitary gland may play a role in these effects.

  19. Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate

    SciTech Connect

    Moffit, Jeffrey S.; Koza-Taylor, Petra H.; Holland, Ricky D.; Thibodeau, Michael S.; Beger, Richard D.; Lawton, Michael P.; Manautou, Jose E. . E-mail: jose.manautou@uconn.edu

    2007-07-15

    Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPAR{alpha}) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPAR{alpha}-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPAR{alpha}-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring (registered) software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

  20. Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse

    PubMed Central

    Fon Tacer, Klementina; Bookout, Angie L.; Ding, Xunshan; Kurosu, Hiroshi; John, George B.; Wang, Lei; Goetz, Regina; Mohammadi, Moosa; Kuro-o, Makoto; Mangelsdorf, David J.; Kliewer, Steven A.

    2010-01-01

    Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals. PMID:20667984

  1. Gene expression profiles of Spo11-/- mouse testes with spermatocytes arrested in meiotic prophase I.

    PubMed

    Smirnova, Natalya A; Romanienko, Peter J; Khil, Pavel P; Camerini-Otero, R Daniel

    2006-07-01

    Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11(-/ -)adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11(-/ -)and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.

  2. Haloperoxidase Mediated Quorum Quenching by Nitzschia cf pellucida: Study of the Metabolization of N-Acyl Homoserine Lactones by a Benthic Diatom

    PubMed Central

    Syrpas, Michail; Ruysbergh, Ewout; Blommaert, Lander; Vanelslander, Bart; Sabbe, Koen; Vyverman, Wim; De Kimpe, Norbert; Mangelinckx, Sven

    2014-01-01

    Diatoms are known to produce a variety of halogenated compounds, which were recently shown to have a role in allelopathic interactions between competing species. The production of these compounds is linked to haloperoxidase activity. This research, has shown that this system may also be involved in diatom-bacteria interactions via the H2O2 dependent inactivation of a type of quorum sensing (QS) molecule, i.e., N-β-ketoacylated homoserine lactones (AHLs), by a natural haloperoxidase system from the benthic diatom Nitzschia cf pellucida. The AHL degradation pathway towards corresponding halogenated derivatives was elucidated via HPLC-MS analysis and the synthesis of a broad series of novel halogenated AHL analogues as reference compounds. Furthermore, their biological activity as quorum sensing modulators was directly compared and evaluated against a series of naturally occurring β-keto-AHLs. It has been demonstrated that the loss of the QS activity results from the final cleavage of the halogenated N-acyl chain of the signal molecules. PMID:24445305

  3. Complete genome sequencing of Pandoraea pnomenusa RB38 and Molecular Characterization of Its N-acyl homoserine lactone synthase gene ppnI

    PubMed Central

    Lim, Yan-Lue; Ee, Robson; How, Kah-Yan; Lee, Siew-Kim; Yong, Delicia; Tee, Kok Keng; Yin, Wai-Fong

    2015-01-01

    In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea. PMID:26336650

  4. N-Acyl Homoserine Lactone-Mediated Quorum Sensing with Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control

    PubMed Central

    Paul, Diby

    2014-01-01

    Membrane biofouling remains a severe problem to be addressed in wastewater treatment systems affecting reactor performance and economy. The finding that many wastewater bacteria rely on N-acyl homoserine lactone-mediated quorum sensing to synchronize their activities essential for biofilm formations; the quenching bacterial quorum sensing suggests a promising approach for control of membrane biofouling. A variety of quorum quenching compounds of both synthetic and natural origin have been identified and found effective in inhibition of membrane biofouling with much less environmental impact than traditional antimicrobials. Work over the past few years has demonstrated that enzymatic quorum quenching mechanisms are widely conserved in several prokaryotic organisms and can be utilized as a potent tool for inhibition of membrane biofouling. Such naturally occurring bacterial quorum quenching mechanisms also play important roles in microbe-microbe interactions and have been used to develop sustainable nonantibiotic antifouling strategies. Advances in membrane fabrication and bacteria entrapment techniques have allowed the implication of such quorum quenching bacteria for better design of membrane bioreactor with improved antibiofouling efficacies. In view of this, the present paper is designed to review and discuss the recent developments in control of membrane biofouling with special emphasis on quorum quenching bacteria that are applied in membrane bioreactors. PMID:25147787

  5. N-Methyltaurine N-acyl amidated bile acids and deoxycholic acid in the bile of angelfish (Pomacanthidae): a novel bile acid profile in Perciform fish.

    PubMed

    Satoh Née Okihara, Rika; Saito, Tetsuya; Ogata, Hiroaki; Ohsaki, Ayumi; Iida, Takashi; Asahina, Kiyoshi; Mitamura, Kuniko; Ikegawa, Shigeo; Hofmann, Alan F; Hagey, Lee R

    2014-02-01

    Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack.

  6. N-acylated alanine methyl esters (NAMEs) from Roseovarius tolerans, structural analogs of quorum-sensing autoinducers, N-acylhomoserine lactones.

    PubMed

    Bruns, Hilke; Thiel, Verena; Voget, Sonja; Patzelt, Diana; Daniel, Rolf; Wagner-Döbler, Irene; Schulz, Stefan

    2013-09-01

    The Roseobacter clade is one of the most important bacteria group living in the ocean. Liquid cultures of Roseovarius tolerans EL 164 were investigated for the production of autoinducers such as N-acylhomoserine lactones (AHLs) and other secondary metabolites. The XAD extracts were analyzed by GC/MS. Two AHLs, Z7-C14 : 1-homoserine lactone (HSL) and C15 : 1-HSL, were identified. Additionally, the extract contained five compounds with molecular-ion peaks at m/z 104, 145, and 158, thus exhibiting mass spectra similar to those of AHLs with corresponding peaks at m/z 102, 143, and 156. Isolation of the main compound by column chromatography, NMR analysis, dimethyl disulfide derivatization for the determination of the location of the CC bond and finally synthesis of the compound with the proposed structure confirmed the compound to be (Z)-N-(hexadec-9-enoyl)alanine methyl ester. Four additional minor compounds were identified as C14 : 0-, C15 : 0-, C16 : 0-, and C17 : 1-N-acylated alanine methyl esters (NAMEs). All NAMEs have not been described from natural sources before. A BLASTp search showed the presence of AHL-producing luxI genes, but no homologous genes potentially responsible for the structurally closely related NAMEs were found. The involvement of the NAMEs in chemical communication processes of the bacteria is discussed.

  7. Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants.

    PubMed

    Götz, Christine; Fekete, Agnes; Gebefuegi, Istvan; Forczek, Sándor T; Fuksová, Kvetoslava; Li, Xiaojing; Englmann, Matthias; Gryndler, Milan; Hartmann, Anton; Matucha, Miroslav; Schmitt-Kopplin, Philippe; Schröder, Peter

    2007-11-01

    Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.

  8. N-Acylated chitosan bis(arylcarbamate)s: A class of promising chiral separation materials with powerful enantioseparation capability and high eluents tolerability.

    PubMed

    Tang, Sheng; Liu, Jian-De; Bin, Qin; Fu, Ke-Qin; Wang, Xiao-Chen; Luo, Ying-Bin; Huang, Shao-Hua; Bai, Zheng-Wu

    2016-12-09

    In order to comprehensively understand the influence of coordination of the substituent at 2-position with those at 3- and 6-positions on the properties of chitosan derivatives, a series of chitosan 3,6-bis(arylcarbamate)-2-(amide)s (CACAs) and the related chiral stationary phases (CSPs) were prepared and reported in the present study. Specifically, chitosan was N-acylated with carboxylic acid anhydrides, and then further derivatized with various aryl isocyanates to afford CACAs, from which a class of coated-type CSPs were prepared. When the substituent introduced on the acyl group at 2-position and those on the phenyl group of the carbamates at 3- and 6-positions were fittingly combined, these prepared CACAs based CSPs would exhibit powerful chiral recognition ability, further resulting in a class of promising chiral separation materials with excellent enantioseparation performance. Meanwhile, these newly developed materials with suitable molecular weight also bear a high tolerability towards organic solvents, even including pure tetrahydrofuran, thus broadening their application in enantiomeric separation.

  9. The type III neurofilament peripherin is expressed in the tuberomammillary neurons of the mouse

    PubMed Central

    Eriksson, Krister S; Zhang, Shengwen; Lin, Ling; Larivière, Roxanne C; Julien, Jean-Pierre; Mignot, Emmanuel

    2008-01-01

    Background Peripherin, a type III neuronal intermediate filament, is widely expressed in neurons of the peripheral nervous system and in selected central nervous system hindbrain areas with projections towards peripheral structures, such as cranial nerves and spinal cord neurons. Peripherin appears to play a role in neurite elongation during development and axonal regeneration, but its exact function is not known. We noticed high peripherin expression in the posterior hypothalamus of mice, and decided to investigate further the exact location of expression and function of peripherin in the mouse posterior hypothalamus. Results In situ hybridization indicated expression of peripherin in neurons with a distribution reminiscent of the histaminergic neurons, with little signal in any other part of the forebrain. Immunocytochemical staining for histidine decarboxylase and peripherin revealed extensive colocalization, showing that peripherin is produced by histaminergic neurons in all parts of the tuberomammillary nucleus. We next used histamine immunostaining in peripherin knockout, overexpressing and wild type mice to study if altered peripherin expression affects these neurons, but could not detect any visible difference in the appearance of these neurons or their axons. Peripherin knockout mice and heterozygotic littermates were used for measurement of locomotor activity, feeding, drinking, and energy expenditure. Both genotypes displayed diurnal rhythms with all the parameters higher during the dark period. The respiratory quotient, an indicator of the type of substrate being utilized, also exhibited a significant diurnal rhythm in both genotypes. The diurnal patterns and the average values of all the recorded parameters for 24 h, daytime and night time were not significantly different between the genotypes, however. Conclusion In conclusion, we have shown that peripherin is expressed in the tuberomammillary neurons of the mouse hypothalamus. Monitoring of locomotor

  10. Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa.

    PubMed

    Nayernia, Karim; Diaconu, Mihaela; Aumüller, Gerhard; Wennemuth, Gunther; Schwandt, Iris; Kleene, Kenneth; Kuehn, Hartmut; Engel, Wolfgang

    2004-04-01

    Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.

  11. Analysis of transcription factor Stk40 expression and function during mouse pre-implantation embryonic development.

    PubMed

    Zhang, Junqiang; Zhang, Juanjuan; Zhao, Chun; Shen, Rong; Guo, Xirong; Li, Chaojun; Ling, Xiufeng; Liu, Chang

    2014-02-01

    Determining the molecular mechanisms in the regulation of early embryonic development is crucial for assisted reproductive technology clinical applications. Serine/threonine protein kinase 40 (Stk40) is a member of the serine/threonine kinase family. It is essential in diverse signaling pathways associated with a wide range of cellular activities, including proliferation, differentiation, survival and apoptosis. However, its involvement and molecular mechanisms in pre‑implantation embryonic development have not been well‑defined. In the present study, it was demonstrated that Stk40 was involved in the development of mouse pre‑implantation embryos. Immunofluorescence and confocal microscopy analyses showed that Stk40 was equally expressed in the nuclei and cytoplasm during all stages of pre‑implantation mouse embryos of imprinting control region mice. Reverse transcription‑polymerase chain reaction showed a significantly higher transcription rate of Stk40 mRNA in the two‑cell stage. The results demonstrated that Stk40 downregulation by microinjection of small interfering RNA into the mouse zygote markedly decreased the blastulation compared with that in the control (Stk40i‑1 vs. control: 65.2% and 77.0%, P<0.05 and Stk40i‑2 vs. control: 49.8% and 70.1%, respectively, P<0.05). In addition, silencing of Stk40 significantly increased the transcription rate of reticulocalbin‑2, whereas that of the homeobox protein, Cdx2, was decreased. In conclusion, the results suggested that Stk40 may be critical in the development of pre‑implantation embryos.

  12. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

    PubMed Central

    Elima, K; Eerola, I; Rosati, R; Metsäranta, M; Garofalo, S; Perälä, M; De Crombrugghe, B; Vuorio, E

    1993-01-01

    Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice. Images Figure 3 Figure 5 Figure 6 PMID:8424763

  13. Distinct expression of Cbln family mRNAs in developing and adult mouse brains.

    PubMed

    Miura, Eriko; Iijima, Takatoshi; Yuzaki, Michisuke; Watanabe, Masahiko

    2006-08-01

    Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family.

  14. Characterization of dopamine D1 and D2 receptor-expressing neurons in the mouse hippocampus.

    PubMed

    Gangarossa, Giuseppe; Longueville, Sophie; De Bundel, Dimitri; Perroy, Julie; Hervé, Denis; Girault, Jean-Antoine; Valjent, Emmanuel

    2012-12-01

    The hippocampal formation is part of an anatomical system critically involved in learning and memory. Increasing evidence suggests that dopamine plays an important role in learning and memory as well as in several forms of synaptic plasticity. However, the precise identification of neuronal populations expressing D1 or D2 dopamine receptors within the hippocampus is still lacking. To clarify this issue, we used BAC transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter of dopamine D1 or D2 receptors. In Drd1a-EGFP mice, sparse GFP-expressing neurons were detected among glutamatergic projecting neurons of the granular layer of the dentate gyrus and GABAergic interneurons located in the hilus. A dense immunofluorescence was observed in the outer and medial part of the molecular layer of the dentate gyrus as well as in the inner part of the molecular layer of CA1 corresponding to the terminals of pyramidal neurons of the entorhinal cortex defining the perforant and the temporo-ammonic pathway respectively. Finally, scattered D1 receptor-expressing neurons were also identified as GABAergic interneurons in the CA3/CA1 fields of the hippocampus. In Drd2-EGFP transgenic mice, GFP was exclusively detected in the glutamatergic mossy cells located in the polymorphic layer of the dentate gyrus. This pattern was confirmed in Drd2-Cre mice crossed with NLS-LacZ-Tau(mGFP) :LoxP and RCE:LoxP reporter lines. Our results demonstrate that D1 and D2 receptor-expressing neurons are strictly segregated in the mouse hippocampus. By clarifying the identity of D1 and D2 receptor-expressing neurons in the hippocampus, this study establishes a basis for future investigations aiming at elucidating their roles in the hippocampal network.

  15. Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Laskin, Debra L. Heck, Diane E.; Laskin, Jeffrey D.

    2008-09-15

    Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.

  16. Embryonic and Postnatal Expression of Aryl Hydrocarbon Receptor mRNA in Mouse Brain

    PubMed Central

    Kimura, Eiki; Tohyama, Chiharu

    2017-01-01

    Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situ hybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain. PMID:28223923

  17. Dynamic expression of retinoic acid synthesizing and metabolizing enzymes in the developing mouse inner ear

    PubMed Central

    Romand, Raymond; Kondo, Takako; Fraulob, Valérie; Petkovich, Martin; Dollé, Pascal; Hashino, Eri

    2008-01-01

    Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatio-temporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3 and 4 (Raldh1, Raldh2, Raldh3 and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear using quantitative RT-PCR, in situ hybridization and Western blot analysis. Quantitative RT-PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages, but that Cyp26 expression was down-regulated during embryonic development. Conversely, the expression levels of Raldh2 and -3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, while Raldh2 was present in the vestibular end organ. At early embryonic stages as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, while the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3 and Cyp26B1 were confined to the stria vascularis, spiral ganglion and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggests tissue changes in the sensitivity to retinoic acid concentrations. PMID:16615129

  18. Prominent expression of xenobiotic efflux transporters in mouse extraembryonic fetal membranes compared with placenta.

    PubMed

    Aleksunes, Lauren M; Cui, Yue; Klaassen, Curtis D

    2008-09-01

    Fetal exposure to xenobiotics can be restricted by transporters at the interface between maternal and fetal circulation. Previous work identified transporters in the placenta; however, less is known about the presence of these transporters in the fetal membranes (i.e., yolk sac and amniotic membranes). The purpose of this study was to quantify mRNA and protein expression of xenobiotic transporters in mouse placenta and fetal membranes during mid to late gestation. Concepti (placenta and fetal membranes, gestation day 11) or placenta and fetal membranes (gestation days 14 and 17) were collected from pregnant mice and analyzed for expression of multidrug resistance-associated proteins (Mrps), multidrug resistance proteins (Mdrs), multidrug and toxin extrusion proteins (Mates), breast cancer resistance protein (Bcrp), and organic anion-transporting polypeptides (Oatps). Maternal liver and kidneys were also collected at day 14 for mRNA and immunohistochemical analysis. mRNA expression of Mrp, Mdr, Bcrp, Mate-1, and Oatp isoforms was detected at day 11. The uptake carriers Oatp2a1, 3a1, 4a1, and 5a1 showed placenta-predominant expression. At days 14 and 17, fetal membranes expressed higher mRNA levels of the efflux transporters Mrp2 (7-fold), Mrp4 (5-fold), Mrp5 (3-fold), Mrp6 (12-fold), Bcrp (2-fold), and Mate-1 (7-fold) than placenta. Western blot analysis of Mrp2, Mrp4, Mrp6, and Bcrp confirmed higher expression in fetal membranes. Immunostaining revealed apical (Mrp2 and Bcrp) and basolateral (Mrp4, 5, and 6) cellular localization in epithelial cells of the yolk sac. In conclusion, xenobiotic transporters in the fetal membranes may provide an additional route to protect the fetus against endogenous chemicals and xenobiotics.

  19. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

    PubMed Central

    Bai, Lixia; Rohrschneider, Larry R.

    2010-01-01

    Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP+ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP+ cells can regenerate the mammary epithelial network. GFP+ mammary epithelial cells are p63+, CD24mod, CD49fhigh, and CD29high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP+ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells. PMID:20810647

  20. Mouse lymphomyeloid cells can function with significantly decreased expression levels of cytochrome C.

    PubMed

    Shilov, E S; Kislyakov, I V; Gorshkova, E A; Zvartsev, R V; Drutskaya, M S; Mufazalov, I A; Skulachev, V P; Nedospasov, S A

    2014-12-01

    Cytochrome c is an indispensable electron carrier in the mitochondrial respiratory chain and also an important mediator of the internal pathway triggering apoptosis. Mice with a complete deficiency of the Cycs gene encoding the somatic cytochrome c die during the embryogenesis. Using the technology of LoxP-cre-dependent tissue-specific recombination, we obtained some mouse strains with significantly reduced expression of cytochrome c in certain cell types ("conditional genetic knockdown"). This knockdown was achieved by abrogation of the normal splicing of the Cycs locus pre-mRNA due to an additional acceptor site inside the stop-cassette neo(r). Previously, we observed embryonic lethality in homozygous mice with the same knockdown of cytochrome c in all cells of the organism. In the present work we studied two novel mouse strains with conditional knockdown of the Cycs gene in T lymphocytes and macrophages. Somewhat surprisingly, the mice of these two strains under normal conditions were not phenotypically different from the wild-type mice, either on the whole organism level or on the level of activity of individual target cells. Thus, the amount of cytochrome c in lymphomyeloid cells does not affect their development and normal functioning.

  1. Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells.

    PubMed

    Rabiee, Farzaneh; Forouzanfar, Mahboobeh; Ghazvini Zadegan, Faezeh; Tanhaei, Somayeh; Ghaedi, Kamran; Motovali Bashi, Majid; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2014-11-10

    Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.

  2. Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

    PubMed Central

    Petrunia, Igor V.; Frolova, Olga Y.; Komarova, Tatiana V.; Kiselev, Sergey L.; Citovsky, Vitaly; Dorokhov, Yuri L.

    2008-01-01

    Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectible levels of genetic transformation of mouse organs. PMID:18523638

  3. Catecholamines are required for androgen-induced ODC expression but not for hypertrophy of mouse kidney.

    PubMed

    Manteuffel-Cymborowska, M; Peska, M; Chmurzyńska, W; Grzelakowska-Sztabert, B

    1997-05-27

    Catecholamine depletion, evoked by reserpine, dramatically impaired (5-fold) the testosterone-induced increase of ornithine decarboxylase (ODC) activity in female mouse kidney. However, reserpine did not prevent kidney hypertrophy evoked by testosterone. This is evidenced by the activity of sensitive, biochemical markers of renal hypertrophy, namely arginase and ornithine aminotransferase (OAT), that responded with the increase and decrease of activities to testosterone treatment, respectively. Arginine and ornithine, substrates and/or products of marker enzymes, showed a striking homeostasis as their level was not affected by testosterone and reserpine, and only slightly by DFMO. Northern blot analysis revealed that the ODC mRNA level, that was increased 10-fold by testosterone, was decreased 2-fold in catecholamine-depleted hypertrophic kidney. Thus, ODC transcript level, lowered by reserpine, correlated partially with an attenuated response of ODC activity to testosterone. This was in contrast to DFMO, which inhibited ODC activity, but significantly increased its mRNA content. It is concluded that catecholamines could be involved together with testosterone in regulation of the ODC gene expression in mouse kidney.

  4. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

    PubMed

    Chao, M V; Mellon, P; Charnay, P; Maniatis, T; Axel, R

    1983-02-01

    We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

  5. Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld.

    PubMed Central

    Evans, G A; Margulies, D H; Camerini-Otero, R D; Ozato, K; Seidman, J G

    1982-01-01

    A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism. PMID:6952248

  6. Expression and function of channelrhodopsin 2 in mouse outer hair cells

    NASA Astrophysics Data System (ADS)

    Chen, Fangyi; Wu, Tao; Wilson, Teresa; Subhash, Hrebesh; Omelchenko, Irina; Bateschell, Michael; Wang, Lingyan; Brigande, John; Jiang, Zhi-Gen; Nuttall, Alfred

    2013-03-01

    Outer hair cell (OHC) is widely accepted as the origin of cochlear amplification, a mechanism that accounts for the extreme sensitivity of the mammalian hearing. The key process of cochlear amplification is the reverse transduction, where the OHC changes its length under electrical stimulation. In this study, we developed a method to modulate electro-mechanical transduction with an optogenetic approach based on channelrhodopsin 2 (ChR2), a direct lightactivated non-selective cation channel (NSCC). We specifically expressed ChR2 in mouse cochlea OHCs through in uterus injection of adenovirus vector with ChR2 in fusion with the fluorescent marker tdTomato. We also transfected ChR2(H134R), a point mutant of ChR2, with plasmid to an auditory cell line (HEI-OC1). With whole cell recording, we found that blue light (470 nm) elicited a current with a reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types.

  7. Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

    PubMed Central

    QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

    2016-01-01

    Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

  8. Profiles of gene expression changes in L5178Y mouse lymphoma cells treated with methyl methanesulfonate and sodium chloride.

    PubMed

    Seidel, Shawn D; Sparrow, Barney R; Kan, H Lynn; Stott, William T; Schisler, Melissa R; Linscombe, V Ann; Gollapudi, B Bhaskar

    2004-05-01

    Treatment of cells with genotoxic chemicals is expected to set into motion a series of events including gene expression changes to cope with the damage. We have investigated gene expression changes in L5178Y TK(+/-) mouse lymphoma cells in culture following treatment with methyl methanesulfonate (MMS), a direct acting genotoxin, and sodium chloride (NaCl), which induces mutations in these cells through indirect mechanisms at high concentrations. The mouse lymphoma cells were treated for 4 or 24 h and the cells were harvested for RNA isolation at the end of the treatment. Analysis of the transcriptome was performed using Clontech Mouse 1.2K cDNA microarrays (1185 genes) and hybridized using 32P-labeled cDNA. The microwell methodology was used to quantify the mutagenic response. Of the genes examined, MMS altered the expression (1.5-fold or more) of only five (four at 4 h and one after 24 h treatment). NaCl altered two genes after 4 h treatment, but after 24 h it altered 19 genes (13 down- and six up-regulated). Both compounds altered the expression of several genes associated with apoptosis and NaCl altered genes involved in DNA damage/response and GTP-related proteins. This, along with other data, indicates that the widely used L5178Y TK(+/-) mouse lymphoma cells in culture are relatively recalcitrant in terms of modulating gene expression to deal with genotoxic insult.

  9. In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm.

    PubMed

    Giritharan, G; Delle Piane, L; Donjacour, A; Esteban, F J; Horcajadas, J A; Maltepe, E; Rinaudo, P

    2012-03-01

    Differences in gene expression and imprinting have been reported, comparing in vivo versus in vitro generated preimplantation embryos. Furthermore, mouse studies have shown that placenta development is altered following in vitro culture. However, the molecular mechanisms underlying these findings are unknown. We therefore isolated trophectoderm (TE) and inner cell mass (ICM) cells from in vivo and in vitro fertilization (IVF) embryos and evaluated their transcriptome using microarrays. We found that the transcriptomes of in vitro produced ICM and TE cells showed remarkably few differences compared to ICM and TE cells of in vivo generated embryos. In vitro fertilization embryos showed a reduced number of TE cells compared to in vivo embryos. In addition, TE of IVF embryos showed significant downregulation of solute transporter genes and of genes involved in placenta formation (Eomesodermin, Socs3) or implantation (Hbegf). In summary, IVF and embryo culture significantly affects the transcriptome of ICM and TE cells.

  10. In Vitro Culture of Mouse Embryos Reduces Differential Gene Expression Between Inner Cell Mass and Trophectoderm

    PubMed Central

    Giritharan, G.; Piane, L. Delle; Donjacour, A.; Esteban, F. J.; Horcajadas, J. A.; Maltepe, E.; Rinaudo, P.

    2012-01-01

    Differences in gene expression and imprinting have been reported, comparing in vivo versus in vitro generated preimplantation embryos. Furthermore, mouse studies have shown that placenta development is altered following in vitro culture. However, the molecular mechanisms underlying these findings are unknown. We therefore isolated trophectoderm (TE) and inner cell mass (ICM) cells from in vivo and in vitro fertilization (IVF) embryos and evaluated their transcriptome using microarrays. We found that the transcriptomes of in vitro produced ICM and TE cells showed remarkably few differences compared to ICM and TE cells of in vivo generated embryos. In vitro fertilization embryos showed a reduced number of TE cells compared to in vivo embryos. In addition, TE of IVF embryos showed significant downregulation of solute transporter genes and of genes involved in placenta formation (Eomesodermin, Socs3) or implantation (Hbegf). In summary, IVF and embryo culture significantly affects the transcriptome of ICM and TE cells. PMID:22383776

  11. Allotopic expression of ATP6 in the mouse as a transgenic model of mitochondrial disease.

    PubMed

    Dunn, David A; Pinkert, Carl A

    2015-01-01

    Progress in animal modeling of polymorphisms and mutations in mitochondrial DNA (mtDNA) is not as developed as nuclear transgenesis due to a host of cellular and physiological distinctions. mtDNA mutation modeling is of critical importance as mutations in the mitochondrial genome give rise to a variety of pathological conditions and play a contributing role in many others. Nuclear localization and transcription of mtDNA genes followed by cytoplasmic translation and transport into mitochondria (allotopic expression, AE) provide an opportunity to create in vivo modeling of a targeted mutation in mitochondrial genes and has been suggested as a strategy for gene replacement therapy in patients harboring mitochondrial DNA mutations. Here, we use our AE approach to transgenic mouse modeling of the pathogenic human T8993G mutation in mtATP6 as a case study for designing AE animal models.

  12. Synthesis of 3-tetrazolylmethyl-azepino[4,5-b]indol-4-ones in two reaction steps: (Ugi-azide/N-acylation/SN2)/free radical cyclization and docking studies to a 5-Ht(6) model.

    PubMed

    Gordillo-Cruz, Raul E; Rentería-Gómez, Angel; Islas-Jácome, Alejandro; Cortes-García, Carlos J; Díaz-Cervantes, Erik; Robles, Juvencio; Gámez-Montaño, Rocío

    2013-10-14

    A series of nine novel 3-tetrazolylmethyl-azepino[4,5-b]indol-4-ones were prepared in moderate to good overall yields in only two reaction steps. The first step consisted of a one-pot sequential process of an Ugi-azide multicomponent reaction, N-acylation and SN2 to give the xanthates. The second step was an intramolecular cyclization under free radical conditions. Also, their binding modes have been modelled using docking techniques.

  13. Differential Hippocampal Gene Expression and Pathway Analysis in an Etiology-Based Mouse Model of Major Depressive Disorder

    PubMed Central

    Zubenko, George S.; Hughes, Hugh B.; Jordan, Rick M.; Lyons-Weiler, James; Cohen, Bruce M.

    2015-01-01

    We have recently reported the creation and initial characterization of an etiology-based recombinant mouse model of a severe and inherited form of Major Depressive Disorder (MDD). This was achieved by replacing the corresponding mouse DNA sequence witha6-base DNA sequence from the human CREB1promoterthat is associated with MDD in individuals from families with recurrent, early-onset MDD (RE-MDD). In the current study, we explored the effect of the pathogenic Creb1 allele on gene expression in the mouse hippocampus, a brain region that is altered in structure and function in MDD. Mouse whole-genome profiling was performed using the Illumina MouseWG-6 v2.0 Expression BeadChip microarray. Univariate analysis identified 269 differentially-expressed genes in the hippocampus of the mutant mouse. Pathway analyses highlighted 11 KEGG pathways: the phosphatidylinositol signaling system, which has been widely implicated in MDD, Bipolar Disorder, and the action of mood stabilizers; gap junction and long-term potentiation, which mediate cognition and memory functions often impaired in MDD; cardiac muscle contraction, insulin signaling pathway, and three neurodegenerative brain disorders (Alzheimer’s, Parkinson’s, and Huntington’s Diseases) that are associated with MDD; ribosome and proteasome pathways affecting protein synthesis/degradation; and the oxidative phosphorylation pathway that is key to energy production. These findings illustrate the merit of this congenic C57BL/6 recombinant mouse as a model of RE-MDD, and demonstrate its potential for highlighting molecular and cellular pathways that contribute to the biology of MDD. The results also inform our understanding of the mechanisms that underlie the comorbidity of MDD with other disorders. PMID:25059218

  14. Neuroligin 2 is expressed in synapses established by cholinergic cells in the mouse brain.

    PubMed

    Takács, Virág T; Freund, Tamás F; Nyiri, Gábor

    2013-01-01

    Neuroligin 2 is a postsynaptic protein that plays a critical role in the maturation and proper function of GABAergic synapses. Previous studies demonstrated that deletion of neuroligin 2 impaired GABAergic synaptic transmission, whereas its overexpression caused increased inhibition, which suggest that its presence strongly influences synaptic function. Interestingly, the overexpressing transgenic mouse line showed increased anxiety-like behavior and other behavioral phenotypes, not easily explained by an otherwise strengthened GABAergic transmission. This suggested that other, non-GABAergic synapses may also express neuroligin 2. Here, we tested the presence of neuroligin 2 at synapses established by cholinergic neurons in the mouse brain using serial electron microscopic sections double labeled for neuroligin 2 and choline acetyltransferase. We found that besides GABAergic synapses, neuroligin 2 is also present in the postsynaptic membrane of cholinergic synapses in all investigated brain areas (including dorsal hippocampus, somatosensory and medial prefrontal cortices, caudate putamen, basolateral amygdala, centrolateral thalamic nucleus, medial septum, vertical- and horizontal limbs of the diagonal band of Broca, substantia innominata and ventral pallidum). In the hippocampus, the density of neuroligin 2 labeling was similar in GABAergic and cholinergic synapses. Moreover, several cholinergic contact sites that were strongly labeled with neuroligin 2 did not resemble typical synapses, suggesting that cholinergic axons form more synaptic connections than it was recognized previously. We showed that cholinergic cells themselves also express neuroligin 2 in a subset of their input synapses. These data indicate that mutations in human neuroligin 2 gene and genetic manipulations of neuroligin 2 levels in rodents will potentially cause alterations in the cholinergic system as well, which may also have a profound effect on the functional properties of brain circuits

  15. Gene expression profiling in the lung and liver of PFOA-exposed mouse fetuses.

    PubMed

    Rosen, Mitchell B; Thibodeaux, Julie R; Wood, Carmen R; Zehr, Robert D; Schmid, Judith E; Lau, Christopher

    2007-09-24

    Perfluorooctanoic acid (PFOA) is a stable perfluoroalkyl acid used to synthesize fluoropolymers during the manufacture of a wide variety of products. Concerns have been raised over the potential health effects of PFOA because it is persistent in the environment and can be detected in blood and other tissues of many animal species, including humans. PFOA has also been shown to induce growth deficits and mortality in murine neonates. To better understand the mechanism of PFOA induced developmental toxicity, lung and liver gene expression profiling was conducted in PFOA-exposed full-term mouse fetuses. Thirty timed-pregnant CD-1 mice were orally dosed from gestation days 1-17 with either 0, 1, 3, 5, or 10mg/(kgday) PFOA in water. At term, fetal lung and liver were collected, total RNA prepared, and samples pooled from three fetuses per litter. Five biological replicates consisting of individual litter samples were then evaluated for each treatment group using Affymetrix mouse 430_2 microarrays. The expression of genes related to fatty acid catabolism was altered in both the fetal liver and lung. In the fetal liver, the effects of PFOA were robust and also included genes associated with lipid transport, ketogenesis, glucose metabolism, lipoprotein metabolism, cholesterol biosynthesis, steroid metabolism, bile acid biosynthesis, phospholipid metabolism, retinol metabolism, proteosome activation, and inflammation. These changes are consistent with transactivation of PPARalpha, although, with regard to bile acid biosynthesis and glucose metabolism, non-PPARalpha related effects were suggested as well. Additional studies will be needed to more thoroughly address the role of PPARalpha, and other nuclear receptors, in PFOA mediated developmental toxicity.

  16. The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

    PubMed

    Schoor, M; Schuster-Gossler, K; Gossler, A

    1993-07-01

    Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.

  17. The precerebellar linear nucleus in the mouse defined by connections, immunohistochemistry, and gene expression.

    PubMed

    Fu, YuHong; Tvrdik, Petr; Makki, Nadja; Palombi, Olivier; Machold, Robert; Paxinos, George; Watson, Charles

    2009-05-19

    The linear nucleus (Li) is a prominent cell group in the caudal hindbrain, which was first described in a study of cerebellar afferents in the rat by [Watson, C.R.R., Switzer, R.C. III, 1978. Trigeminal projections to cerebellar tactile areas in the rat origin mainly from N. interpolaris and N. principalis. Neurosci. Lett. 10, 77-82.]. It was named for its elongated appearance in transverse sections. Since this original description in the rat, reference to the nucleus seems to have been largely absent from experimental studies of mammalian precerebellar nuclei. We therefore set out to define the cytoarchitecture, cerebellar connections, and molecular characteristics of Li in the mouse. In coronal Nissl sections at the level of the rostral inferior olive, it consists of two parallel bands of cells joined at their dorsal apex by a further band of cells, making the shape of the Greek capital letter pi. Our three-dimensional reconstruction demonstrated that the nucleus is continuous with the lateral reticular nucleus (LRt) and that the ambiguus nucleus sits inside the arch of Li. Cerebellar horseradish peroxidase injections confirmed that the cells of Li project to cerebellum. We have shown that Li cells express Atoh1 and Wnt1 lineage markers that are known to label the rhombic lip derived precerebellar nuclei. We have examined the relationship of Li cells to a number of molecular markers, and have found that many of the cells express a nonphosphorylated epitope in neurofilament H (SMI 32), a feature they share with the LRt. The mouse Li therefore appears to be a rostrodorsal extension of the LRt.

  18. CD24 expression does not affect dopamine neuronal survival in a mouse model of Parkinson's disease

    PubMed Central

    Hayat, Shaista; Carnwath, Tom; Garas, Shaady; Sleeman, Jonathan P.; Barker, Roger A.

    2017-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative condition that is characterised by the loss of specific populations of neurons in the brain. The mechanisms underlying this selective cell death are unknown but by using laser capture microdissection, the glycoprotein, CD24 has been identified as a potential marker of the populations of cells that are affected in PD. Using in situ hybridization and immunohistochemistry on sections of mouse brain, we confirmed that CD24 is robustly expressed by many of these subsets of cells. To determine if CD24 may have a functional role in PD, we modelled the dopamine cell loss of PD in Cd24 mutant mice using striatal delivery of the neurotoxin 6-OHDA. We found that Cd24 mutant mice have an anatomically normal dopamine system and that this glycoprotein does not modulate the lesion effects of 6-OHDA delivered into the striatum. We then undertook in situ hybridization studies on sections of human brain and found—as in the mouse brain—that CD24 is expressed by many of the subsets of the cells that are vulnerable in PD, but not those of the midbrain dopamine system. Finally, we sought to determine if CD24 is required for the neuroprotective effect of Glial cell-derived neurotrophic factor (GDNF) on the dopaminergic nigrostriatal pathway. Our results indicate that in the absence of CD24, there is a reduction in the protective effects of GDNF on the dopaminergic fibres in the striatum, but no difference in the survival of the cell bodies in the midbrain. While we found no obvious role for CD24 in the normal development and maintenance of the dopaminergic nigrostriatal system in mice, it may have a role in mediating the neuroprotective aspects of GDNF in this system. PMID:28182766

  19. Polymorphic expression of a human superficial bladder tumor antigen defined by mouse monoclonal antibodies.

    PubMed Central

    Fradet, Y; Islam, N; Boucher, L; Parent-Vaugeois, C; Tardif, M

    1987-01-01

    Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up. Images PMID:3313389

  20. Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung

    PubMed Central

    Suarez-Mier, Gabriela B.

    2015-01-01

    Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line. PMID:26442852

  1. Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents.

    PubMed

    Purnama, Agung; Hermawan, Hendra; Champetier, Serge; Mantovani, Diego; Couet, Jacques

    2013-11-01

    Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts - magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe-35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe-35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 μm pore size) containing cytostatic amounts of 3.25 mg ml(-1) of Fe-35 Mn powder, 0.25 mg ml(-1) of pure Mn powder or 5 mg ml(-1) of pure iron powder for 24 h. We then conducted a gene expression profiling study from these cells. Exposure of 3T3 cells to Fe-35 Mn was associated with the up-regulation of 75 genes and down-regulation of 59 genes, while 126 were up-regulated and 76 down-regulated genes in the presence of manganese. No genes were found regulated for the iron powder. When comparing the GEP of 3T3 fibroblasts in the presence of Fe-35 Mn and Mn, 68 up-regulated and 54 down-regulated genes were common. These results were confirmed by quantitative RT-PCR for a subset of these genes. This GEP study could provide clues about the mechanism behind degradation products effects on cells of the Fe-35 Mn alloy and may help in the appraisal of its potential for DMM applications.

  2. Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells

    PubMed Central

    IMAI, Hiroyuki; FUJII, Wataru; KUSAKABE, Ken Takeshi; KISO, Yasuo; KANO, Kiyoshi

    2016-01-01

    Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2–2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication. PMID:27569766

  3. Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo.

    PubMed

    Simon, Claire S; Downes, Damien J; Gosden, Matthew E; Telenius, Jelena; Higgs, Douglas R; Hughes, Jim R; Costello, Ita; Bikoff, Elizabeth K; Robertson, Elizabeth J

    2017-02-07

    The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable and only the VPE is required for optimal Eomes expression in vivo Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions embryonic stem cells (ESC), prior to gene activation. The locus resides in a large (500kb) pre-formed compartment in ESC and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a, and four additional putative enhancers display increased chromatin accessibility in DE associated with Smad2/3 binding coincident with transcriptional activation. In contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.

  4. Effect of Crossing C57BL/6 and FVB Mouse Strains on Basal Cytokine Expression

    PubMed Central

    Szade, Agata; Nowak, Witold N.; Szade, Krzysztof; Gese, Anna; Czypicki, Ryszard; Waś, Halina; Dulak, Józef; Józkowicz, Alicja

    2015-01-01

    C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound. PMID:25834307

  5. Serotonin receptor expression along the dorsal–ventral axis of mouse hippocampus

    PubMed Central

    Tanaka, Kenji F.; Samuels, Benjamin Adam; Hen, René

    2012-01-01

    Using in situ hybridization, we describe, for the first time, the profiles of expression of serotonin receptors (Htr/5-HTR) along the dorsal–ventral axis of mouse hippocampus. cRNA probes for most Htrs, excluding Htr6, were used. All hippocampal subregions and the entorhinal cortex cells providing input into the hippocampus were examined. The study shows that some, but not all, Htrs are expressed in the cells of the hippocampal circuitry. At both the subfield and the cell type levels, a somewhat overlapping pattern is observed. Four serotonin receptors, Htr1a, Htr2a, Htr2c and Htr7, display an expression pattern that changes along the dorsal–ventral axis of the hippocampus. Given the proposed functional differentiation of the hippocampus along its long axis, with the dorsal pole more involved in cognitive functions and the ventral pole more involved in mood and anxiety, our results suggest that serotonin receptors enriched in the ventral pole probably contribute to mood- and anxiety-related behaviours. PMID:22826340

  6. Radiation Dose-Rate Effects on Gene Expression in a Mouse Biodosimetry Model

    PubMed Central

    Paul, Sunirmal; Smilenov, Lubomir B.; Elliston, Carl D.; Amundson, Sally A.

    2015-01-01

    In the event of a nuclear accident or radiological terrorist attack, there will be a pressing need for biodosimetry to triage a large, potentially exposed population and to assign individuals to appropriate treatment. Exposures from fallout are likely, resulting in protracted dose delivery that would, in turn, impact the extent of injury. Biodosimetry approaches that can distinguish such low-dose-rate (LDR) exposures from acute exposures have not yet been developed. In this study, we used the C57BL/6 mouse model in an initial investigation of the impact of low-dose-rate delivery on the transcriptomic response in blood. While a large number of the same genes responded to LDR and acute radiation exposures, for many genes the magnitude of response was lower after LDR exposures. Some genes, however, were differentially expressed (P < 0.001, false discovery rate < 5%) in mice exposed to LDR compared with mice exposed to acute radiation. We identified a set of 164 genes that correctly classified 97% of the samples in this experiment as exposed to acute or LDR radiation using a support vector machine algorithm. Gene expression is a promising approach to radiation biodosimetry, enhanced greatly by this first demonstration of its potential for distinguishing between acute and LDR exposures. Further development of this aspect of radiation biodosimetry, either as part of a complete gene expression biodosimetry test or as an adjunct to other methods, could provide vital triage information in a mass radiological casualty event. PMID:26114327

  7. Properties of doublecortin expressing neurons in the adult mouse dentate gyrus.

    PubMed

    Spampanato, Jay; Sullivan, Robert K; Turpin, Fabrice R; Bartlett, Perry F; Sah, Pankaj

    2012-01-01

    The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses.