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Sample records for cells lacking recg

  1. RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

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    Masaki Odahara

    2015-03-01

    Full Text Available Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

  2. Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes

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    Killen Michael W

    2012-10-01

    Full Text Available Abstract Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.

  3. Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes.

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    Killen, Michael W; Stults, Dawn M; Wilson, William A; Pierce, Andrew J

    2012-10-30

    Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.

  4. RecG Directs DNA Synthesis during Double-Strand Break Repair.

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    Benura Azeroglu

    2016-02-01

    Full Text Available Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300, arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

  5. Resolution of joint molecules by RuvABC and RecG following cleavage of the Escherichia coli chromosome by EcoKI.

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    Laura Wardrope

    Full Text Available DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions.

  6. Characterization of the Neisseria meningitidis Helicase RecG

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    Beyene, Getachew Tesfaye; Balasingham, Seetha V.; Frye, Stephan A.; Namouchi, Amine; Homberset, Håvard; Kalayou, Shewit; Riaz, Tahira

    2016-01-01

    Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant. PMID:27736945

  7. Murine fertilized ovum, blastomere and morula cells lacking SP phenotype

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells and some progenitor cells, and that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate and single nucleus fertilized ova), 2-cell stage and 8-cell stage blastomeres, morulas and blastocysts were isolated and directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere and morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property and independent of the culture condition in vitro, and that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disappeared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype, and that SP phenotype might act as a purification marker for partial stem cells such as some pluripotent embryonic stem cells and multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.

  8. B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

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    J. Chen(Florida State University, Tallahassee, U.S.A.); Trounstine, M; Kurahara, C.; Young, F.; Kuo, C C; Y. Xu; Loring, J.F.; Alt, F W; Huszar, D

    1993-01-01

    We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, ...

  9. Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

    OpenAIRE

    2012-01-01

    Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but...

  10. PROPOSED CARDIAC STEM CELLS DERIVED FROM “CARDIOSPHERES” LACK CARDIOMYOGENIC POTENTIAL

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline

       Recent studies have reported that clinical relevant numbers of cardiac stem cells (CSCs) with cardiomyogenic potential can be obtained from small heart tissue biopsies, by an intrinsic ability of CSCs to form beating cardiospheres (CSs) during ex vivo culture. Such data have provided optimism...... that injuried heart tissue may be repaired by stem cell therapy using autologous CS derived cells, and pre-clinical studies have already been described in literature.    Herein, we established CSs from neonatal rats, and by immunofluorescence, qRT-PCR, and microscopic examination we demonstrated...... to form CSs by themselves. Phenotypically, CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential.    Our data imply that at least the murine cardiosphere model seems unsuitable for enrichment of cardiac stem cells with cardiomyogenic...

  11. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

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    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  12. Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

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    Rodriguez, Pilar [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Barquinero, Joan Francesc [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Duran, Assumpta [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Caballin, Maria Rosa [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Ribas, Montserrat [Servei de Radiofisica i Radioproteccio de l' Hospital de la Santa Creu i Sant Pau, 08025 Barcelona (Spain); Barrios, Leonardo, E-mail: Lleonard.Barrios@uab.cat [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain)

    2009-11-02

    Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of {gamma}-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

  13. Tc17 cells mediate vaccine immunity against lethal fungal pneumonia in immune deficient hosts lacking CD4+ T cells.

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    Som Gowda Nanjappa

    Full Text Available Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4(+ T-cell help, vaccine-induced CD8(+ T cells persist and confer resistance against Blastomyces dermatitidis and Histoplasma capsulatum. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8(+ T cells (Tc17 cells have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4(+ T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.

  14. Tc17 cells mediate vaccine immunity against lethal fungal pneumonia in immune deficient hosts lacking CD4+ T cells.

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    Nanjappa, Som Gowda; Heninger, Erika; Wüthrich, Marcel; Gasper, David Joseph; Klein, Bruce S

    2012-01-01

    Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4(+) T-cell help, vaccine-induced CD8(+) T cells persist and confer resistance against Blastomyces dermatitidis and Histoplasma capsulatum. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8(+) T cells (Tc17 cells) have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4(+) T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.

  15. RecJ, ExoI and RecG are required for genome maintenance but not for generation of genetic diversity by repeat-mediated phase variation in Haemophilus influenzae

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    Kumar, Gaurav A.; Woodhall, Mark R.; Hood, Derek W.; Moxon, E. Richard [Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS (United Kingdom); Bayliss, Christopher D. [Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH (United Kingdom)], E-mail: cdb12@le.ac.uk

    2008-04-02

    High levels of genetic diversity are generated in Haemophilus influenzae populations through DNA repeat-mediated phase variation and recombination with DNA fragments acquired by uptake from the external milieu. Conversely, multiple pathways for maintenance of the genome sequence are encoded in H. influenzae genomes. In Escherichia coli, mutations in single-stranded-DNA exonucleases destabilise tandem DNA repeats whilst inactivation of recG can stabilise repeat tracts. These enzymes also have varying effects on recombination. Deletion mutations were constructed in H. influenzae genes encoding homologs of ExoI, RecJ and RecG whilst ExoVII was refractory to mutation. Inactivation of RecJ and RecG, but not ExoI, increased sensitivity to irradiation with ultraviolet light. An increase in spontaneous mutation rate was not observed in single mutants but only when both RecJ and ExoI were mutated. None of the single- or double-mutations increased or decreased the rates of slippage in tetranucleotide repeat tracts. Furthermore, the exonuclease mutants did not exhibit significant defects in horizontal gene transfer. We conclude that RecJ, ExoI and RecG are required for maintenance of the H. influenzae genome but none of these enzymes influence the generation of genetic diversity through mutations in the tetranucleotide repeat tracts of this species.

  16. Characterization of spermatogonial stem cells lacking intercellular bridges and genetic replacement of a mutation in spermatogonial stem cells.

    Directory of Open Access Journals (Sweden)

    Naoki Iwamori

    Full Text Available Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14 null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14(+/- spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.

  17. Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

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    Iwamori, Naoki; Iwamori, Tokuko; Matzuk, Martin M.

    2012-01-01

    Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term. PMID:22719986

  18. How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules?

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    Ole Audun Werner Haabeth

    2014-04-01

    Full Text Available CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor specific antigen by host antigen presenting cells (APCs appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315, where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-g stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed.

  19. LACK OF HETEROTRANSPLANTATION OF MAREK'S DISEASE LYMPHOMA-DERIVED CELL LINES AND MD LYMPHOMA CELLS TO NUDE MICE

    OpenAIRE

    1980-01-01

    Nude mice of BALB/c background were used for the heterotransplantation of Marek's Disease (MD) lymphoma-derived cell lines (MDCC-MSB 1,MDCC-RP 1 and MDCC-JP 2) or MD lymphoma developed in a Marek's disease virus-inoculated chicken. None out of the 57 nude mice developed tumors at the site of inoculation. These nude mice formed cytotoxic antibody against MD lymphoma-derived line cells 6-14 weeks after inoculation. The lack of heterotransplantation of cells from avian origin into nude mice is d...

  20. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α

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    Elizabeth K Lucas

    2015-01-01

    Full Text Available Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α -/- mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV, a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2, structural (neurofilament heavy chain, Nefh, and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1 functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α -/- mice. We observed a significant loss of Purkinje cells by six weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α’s actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency.

  1. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α

    Science.gov (United States)

    Lucas, Elizabeth K.; Reid, Courtney S.; McMeekin, Laura J.; Dougherty, Sarah E.; Floyd, Candace L.; Cowell, Rita M.

    2014-01-01

    Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α) have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α−/− mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV), a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2), structural (neurofilament heavy chain, Nefh), and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1) functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α−/− mice. We observed a significant loss of Purkinje cells by 6 weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α's actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency. PMID

  2. Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

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    Vukovic, Milica; Sepulveda, Catarina; Subramani, Chithra; Guitart, Amélie V; Mohr, Jasmine; Allen, Lewis; Panagopoulou, Theano I; Paris, Jasmin; Lawson, Hannah; Villacreces, Arnaud; Armesilla-Diaz, Alejandro; Gezer, Deniz; Holyoake, Tessa L; Ratcliffe, Peter J; Kranc, Kamil R

    2016-06-09

    The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.

  3. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

    Science.gov (United States)

    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  4. Newly generated cells are increased in hippocampus of adult mice lacking a serine protease inhibitor

    Directory of Open Access Journals (Sweden)

    Sticker Melanie

    2010-06-01

    Full Text Available Abstract Background Neurogenesis in the hippocampal dentate gyrus and the subventricular zone occurs throughout the life of mammals and newly generated neurons can integrate functionally into established neuronal circuits. Neurogenesis levels in the dentate gyrus are modulated by changes in the environment (enrichment, exercise, hippocampal-dependent tasks, NMDA receptor (NMDAR activity, sonic hedgehog (SHH and/or other factors. Results previously, we showed that Protease Nexin-1 (PN-1, a potent serine protease inhibitor, regulates the NMDAR availability and activity as well as SHH signaling. Compared with wild-type (WT, we detected a significant increase in BrdU-labeled cells in the dentate gyrus of mice lacking PN-1 (PN-1 -/- both in controls and after running exercise. Patched homologue 1 (Ptc1 and Gli1 mRNA levels were higher and Gli3 down-regulated in mutant mice under standard conditions and to a lesser extent after running exercise. However, the number of surviving BrdU-positive cells did not differ between WT and PN-1 -/- animals. NMDAR availability was altered in the hippocampus of mutant animals after exercise. Conclusion All together our results indicate that PN-1 controls progenitors proliferation through an effect on the SHH pathway and suggest an influence of the serpin on the survival of newly generated neurons through modulation of NMDAR availability.

  5. Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors

    NARCIS (Netherlands)

    van Es, J.H.; de Geest, N.; van den Born, M.M.W.; Clevers, H.; Hassan, B.A.

    2010-01-01

    Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate into one of the four principal cell types. Signalling pathways, including the Notch signalling pathway, coordinate these differentiation p

  6. Characterization of subpopulation lacking both B-cell and plasma cell markers in Waldenstrom macroglobulinemia cell line.

    Science.gov (United States)

    Wada, Naoki; Zhan, Maosheng; Hori, Yumiko; Honma, Keiichiro; Ikeda, Jun-ichiro; Morii, Eiichi

    2014-01-01

    Cancer cells with tumorigenic potential are limited to a small population known as cancer-initiating cells (CICs). To date, CICs have not been identified in non-Hodgkin's lymphomas. Here, we investigated a candidate of CICs of an indolent non-Hodgkin's lymphoma, Waldenstrom macroglobulinemia (WM), using WM cell line MWCL-1. WM tumor expresses both B-cell and plasma cell markers, CD20 and CD138. When stained with anti-CD20 and anti-CD138 antibodies, MWCL-1 cells were classified into three subpopulations: CD20⁻ CD138⁻, CD20⁺ CD138⁻, and CD20⁺ CD138⁺. When cultured, CD20⁻ CD138⁻ cells yielded all three subpopulations, but CD20⁺ cells did not yield CD20⁻ CD138⁻ cells. Higher reactive oxygen species (ROS) expelling and in vitro colony formation activities were detected in CD20⁻ CD138⁻ cells than in CD20⁺ CD138⁻ and CD20⁺ CD138⁺ cells. When cultured in the absence of serum or with anti-cancer drug, CD20⁻ CD138⁻ cells were resistant to apoptosis. In contrast, CD20⁺ CD138⁺ cells were vulnerable to apoptosis in the same condition. In fact, the immunohistochemical analysis with clinical samples revealed that tumor cells in apoptosis were CD138-positive. The production of all three subpopulations, the efficient ROS expelling and in vitro colony-forming activities, and the resistance to apoptosis suggested that the CD20⁻ CD138⁻ cell might be a candidate of CICs in WM.

  7. Lack of Erythropoietic Inhibitory Effect of Serum From Patients with Congenital Pure Red Cell Aplasia

    Science.gov (United States)

    Geller, Gary; And Others

    1975-01-01

    Serum of five children ages 1 to 19 months with congenital pure red cell aplasia (incomplete or defective development of red blood cells) was injected in normal mice to determine possible inhibition of red blood cell formulating stimulants. (CL)

  8. Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shang-Lang [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chao, Chuck C.-K., E-mail: cckchao@mail.cgu.edu.tw [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Department of Medical Research and Development, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan (China)

    2015-06-16

    A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

  9. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces cell cycle synchronization in different human osteosarcoma cell lines. The UV pulse also has a destabilizing...

  10. A lack of commitment for over 500 million years: conserved animal stem cell pluripotency.

    Science.gov (United States)

    Aboobaker, A Aziz; Kao, Damian

    2012-06-13

    Stem cells, both adult and germline, are the key cells underpinning animal evolution. Yet, surprisingly little is known about the evolution of their shared key feature: pluripotency. Now using genome-wide expression profiling of pluripotent planarian adult stem cells (pASCs), Önal et al (2012) present evidence for deep molecular conservation of pluripotency. They characterise the expression profile of pASCs and identify conserved expression profiles and functions for genes required for mammalian pluripotency. Their analyses suggest that molecular pluripotency mechanisms may be conserved, and tantalisingly that pluripotency in germ stem cells (GSCs) and somatic stem cells (SSCs) may have had shared common evolutionary origins.

  11. Lack of correlation between telomere length and telomerase activity and expression in leukemic cells.

    Science.gov (United States)

    Januszkiewicz, Danuta; Wysoki, Jacek; Lewandowski, Krzysztof; Pernak, Monika; Nowicka, Karina; Rembowska, Jolanta; Nowak, Jerzy

    2003-12-01

    The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).

  12. Lack of p53 Augments Antitumor Functions in Cytolytic T Cells.

    Science.gov (United States)

    Banerjee, Anirban; Thyagarajan, Krishnamurthy; Chatterjee, Shilpak; Chakraborty, Paramita; Kesarwani, Pravin; Soloshchenko, Myroslawa; Al-Hommrani, Mazen; Andrijauskaite, Kristina; Moxley, Kelly; Janakiraman, Harinarayanan; Scheffel, Matthew J; Helke, Kristi; Armenson, Kent; Palanisamy, Viswanathan; Rubinstein, Mark P; Mayer, Elizabeth-Garrett; Cole, David J; Paulos, Chrystal M; Voelkel-Johnson, Christina; Nishimura, Michael I; Mehrotra, Shikhar

    2016-09-15

    Repetitive stimulation of T-cell receptor (TCR) with cognate antigen results in robust proliferation and expansion of the T cells, and also imprints them with replicative senescence signatures. Our previous studies have shown that life-span and antitumor function of T cells can be enhanced by inhibiting reactive oxygen species (ROS) or intervening with ROS-dependent JNK activation that leads to its activation-induced cell death. Because tumor suppressor protein p53 is also a redox active transcription factor that regulates cellular ROS generation that triggers downstream factor-mediating apoptosis, we determined if p53 levels could influence persistence and function of tumor-reactive T cells. Using h3T TCR transgenic mice, with human tyrosinase epitope-reactive T cells developed on p53 knockout (KO) background, we determined its role in regulating antitumor T-cell function. Our data show that as compared with h3T cells, h3T-p53 KO T cells exhibited enhanced glycolytic commitment that correlated with increased proliferation, IFNγ secretion, cytolytic capacity, expression of stemness gene signature, and decreased TGF-β signaling. This increased effector function correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of p53-KO T cells. Pharmacological inhibition of human TCR-transduced T cells using a combination of p53 inhibitors also potentiated the T-cell effector function and improved persistence. Thus, our data highlight the key role of p53 in regulating the tumor-reactive T-cell response and that targeting this pathway could have potential translational significance in adoptive T-cell therapy. Cancer Res; 76(18); 5229-40. ©2016 AACR.

  13. Arrest of Myelination and Reduced Axon Growth when Schwann Cells Lack mTOR

    OpenAIRE

    Sherman, Diane L; Krols, Michiel; Wu, Lai-Man N; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J.

    2012-01-01

    In developing peripheral nerves differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years there has been an increasing understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination together with a growing appreciation of some of the signalling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete....

  14. Smooth muscle cell proliferation in the occluded rat carotid artery: lack of requirement for luminal platelets.

    Science.gov (United States)

    Guyton, J. R.; Karnovsky, M. J.

    1979-01-01

    The relationship of intimal smooth muscle cell proliferation in the permanently occluded rat carotid artery to the presence or absence of luminal platelets was examined. Blood was rinsed from the arterial lumen immediately after occlusion and was replaced by autologous, citrated platelet-rich plasma (PRP, 6 to 20 X 10(5) platelets/microliter) or filtered platelet-poor plasma (PPP, less than 100 platelets/microliter). Occluded arteries were studied after 1 to 28 days by light and electron microscopy. Events occurring within the first 2 days included fibrin clot formation, endothelial degeneration and denudation, transmural migration of polymorphonucelar leukocytes and monocytes, and, in PRP-filled arteries, degranulation and disappearance of platelets. By 7 days a neointima was formed by macrophages and undifferentiated cells. The latter cells had some features of vascular smooth muscle cells and were apparently derived from medial cells which traversed the internal elastic lamina. After 14 days, identifiable smooth muscle cells emerged as the predominant cell type in a rapidly growing intimal plaque. No differences could be discerned between arteries originally filled with PRP or PPP. This experimental model is similar to atherosclerosis in dimensions of avascular area and in coexistence of degenerative, inflammatory, and proliferative processes. Cell proliferation deep within an atherosclerotic plaque could be initiated by factors other than platelets, perhaps by products of inflammatory cells. Images Figure 4 Figure 7 Figure 6 Figure 1 Figure 2 Figure 3 Figure 8 Figure 5 PMID:426040

  15. Lack of functional relevance of isolated cell damage in transplants of Parkinson's disease patients

    DEFF Research Database (Denmark)

    Cooper, Oliver; Astradsson, Arnar; Hallett, Penny;

    2009-01-01

    -like structures. In this review, we discuss why this isolated cell damage is unlikely to affect the overall graft function and how we can use these observations to help us to understand age-related neurodegeneration and refine our future cell replacement therapies....

  16. Arrest of myelination and reduced axon growth when Schwann cells lack mTOR.

    Science.gov (United States)

    Sherman, Diane L; Krols, Michiel; Wu, Lai-Man N; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J

    2012-02-01

    In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.

  17. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...

  18. Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation.

    Science.gov (United States)

    Pasut, Alessandra; Chang, Natasha C; Rodriguez, Uxia Gurriaran; Faulkes, Sharlene; Yin, Hang; Lacaria, Melanie; Ming, Hong; Rudnicki, Michael A

    2016-07-12

    Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7(-/-) satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

  19. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...... tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule....

  20. Proper design of silica nanoparticles combines high brightness, lack of cytotoxicity and efficient cell endocytosis

    Science.gov (United States)

    Rampazzo, Enrico; Voltan, Rebecca; Petrizza, Luca; Zaccheroni, Nelsi; Prodi, Luca; Casciano, Fabio; Zauli, Giorgio; Secchiero, Paola

    2013-08-01

    Silica-based luminescent nanoparticles (SiNPs) show promising prospects in nanomedicine in light of their chemical properties and versatility. In this study, we have characterized silica core-PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in animal models. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2 leukemic cell line and primary normal peripheral blood mononuclear cells) or in adherence (human hepatocarcinoma Huh7 and umbilical vein endothelial cells). Moreover, by multiparametric flow cytometry, we could demonstrate that the highest efficiency of cell uptake and entry was observed with NP-PEG-amino, with a stable persistence of the fluorescence signal associated with SiNPs in the loaded cell populations both in vitro and in vivo settings suggesting this as an innovative method for cell traceability and detection in whole organisms. Finally, experiments performed with the endocytosis inhibitor Genistein clearly suggested the involvement of a caveolae-mediated pathway in SiNP endocytosis. Overall, these data support the safe use of these SiNPs for diagnostic and therapeutic applications.Silica-based luminescent nanoparticles (SiNPs) show promising prospects in nanomedicine in light of their chemical properties and versatility. In this study, we have characterized silica core-PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in animal models. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2

  1. Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance.

    Science.gov (United States)

    Chen, Yanlian; Deng, Zhiqiang; Jiang, Shuai; Hu, Qian; Liu, Haiying; Songyang, Zhou; Ma, Wenbin; Chen, Shi; Zhao, Yong

    2015-01-01

    The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating that telomerase assembly occurs in the absence of Cajal bodies. Moreover, dispersed hTR aggregates and forms foci specifically during S and G2 phase in coilin-KO cells. Colocalization of these hTR foci with telomeres implies proper telomerase trafficking, independent of Cajal bodies. Therefore, telomerase adds similar numbers of TTAGGG repeats to telomeres in coilin-KO and controls cells. Overexpression of TPP1-OB-fold blocks cell cycle-dependent formation of hTR foci and inhibits telomere extension. These findings suggest that telomerase assembly, trafficking and extension occur with normal efficiency in Cajal bodies deficient human cells. Thus, Cajal bodies, as such, are not essential in these processes, although it remains possible that non-coilin components of Cajal bodies and/or telomere binding proteins (e.g. TPP1) do play roles in telomerase biogenesis and telomere homeostasis.

  2. Lack of telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathway

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhen-zhou; XU Ru-xiang; JIANG Xiao-dan; TENG Xiao-hua; LI Gui-tao; ZHOU Yü-xi

    2006-01-01

    Objective: To investigate telomerase activity in rabbit bone marrow stromal cells (BMSCs) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase.Methods: BMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group.No. ZL02134314. 4) supplemented with 10% fetal bovine serum (FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuronspecific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activitys were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA).Results: BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups.Conclusions: Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.

  3. Resistance to mTOR kinase inhibitors in lymphoma cells lacking 4EBP1.

    Directory of Open Access Journals (Sweden)

    Sharmila Mallya

    Full Text Available Inhibitors of the mechanistic target of rapamycin (mTOR hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive "active-site" mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1, a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.

  4. Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction.

    Science.gov (United States)

    Ochocki, Joshua D; Mullen, Daniel G; Wattenberg, Elizabeth V; Distefano, Mark D

    2011-09-01

    Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

  5. Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance

    OpenAIRE

    Chen, Yanlian; Deng, Zhiqiang; Jiang, Shuai; Hu, Qian; Liu, Haiying; Songyang, Zhou; Ma, Wenbin; Chen, Shi; Zhao, Yong

    2014-01-01

    The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating t...

  6. Efficient Human Immunodeficiency Virus (HIV-1) Infection of Cells Lacking PDZD8

    OpenAIRE

    Zhang, Shijian; Sodroski, Joseph

    2015-01-01

    PDZD8 can bind the capsid proteins of different retroviruses, and transient knockdown of PDZD8 results in a decrease in the efficiency of an early, post-entry event in the retrovirus life cycle. Here we used the CRISPR-CAS9 system to create cell lines in which PDZD8 expression is stably eliminated. The PDZD8-knockout cell lines were infected by human immunodeficiency virus (HIV-1) and murine leukemia virus as efficiently as the parental PDZD8-expressing cells. These results indicate that PDZD...

  7. Efficient human immunodeficiency virus (HIV-1) infection of cells lacking PDZD8.

    Science.gov (United States)

    Zhang, Shijian; Sodroski, Joseph

    2015-07-01

    PDZD8 can bind the capsid proteins of different retroviruses, and transient knockdown of PDZD8 results in a decrease in the efficiency of an early, post-entry event in the retrovirus life cycle. Here we used the CRISPR-CAS9 system to create cell lines in which PDZD8 expression is stably eliminated. The PDZD8-knockout cell lines were infected by human immunodeficiency virus (HIV-1) and murine leukemia virus as efficiently as the parental PDZD8-expressing cells. These results indicate that PDZD8 is not absolutely necessary for HIV-1 infection and diminishes its attractiveness as a potential target for intervention.

  8. EFFECTS OF QUERCETIN ON MEMBRANE FLUIDITY OF INJURED VASCULAR ENDOTHELIAL CELLS WITH HYPOXIA AND THE LACK OF GLUCOSE

    Institute of Scientific and Technical Information of China (English)

    林蓉; 刘俊田; 甘伟杰

    2003-01-01

    Objective To study the effects of different concentrations of Quercetin on nitric oxide (NO) production and membrane fluidity of the injured human umbilical vein vascular endothelial cell line(ECV-304) with hypoxia and the lack of glucose. Methods The experiments were performed in the culture of ECV-304 injured with hypoxia and the lack of glucose in vitro. The releases of intracellular lactate dehydrogenase(LDH) of ECV-304 was measured with automatic biochemistry analysis. NO level of ECV-304 was monitored with colorimetry. The membrane fluidity of ECV-304 was measured with the fluorescence polarization method. Results After ECV-304 was cultured in hypoxia and the the lack of glucose for 24 hours, the release of LDH and the membrane fluidity were increased significantly; NO level was decreased. Preincubation of ECV-304 with 20, 80,160μmol*L-1 of Quercetin for 24 hours reduced LDH activity, membrane fluidity and increased the level of NO in hypoxia and the lack of glucose induced ECV-304. Conclusion These results demonstrate that Quercetin can produce the protective effect on hypoxia and the lack of glucose induced injury of ECV-304 by increasing release of NO and changing membrane fluidity.

  9. Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal.

    Science.gov (United States)

    Gradilone, Angela; Raimondi, Cristina; Nicolazzo, Chiara; Petracca, Arianna; Gandini, Orietta; Vincenzi, Bruno; Naso, Giuseppe; Aglianò, Anna Maria; Cortesi, Enrico; Gazzaniga, Paola

    2011-05-01

    Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.

  10. Lack of increased expression of cell surface markers for circulating fibrocyte progenitors in limited scleroderma.

    Science.gov (United States)

    Russo, R; Medbury, H; Guiffre, A; Englert, H; Manolios, N

    2007-07-01

    The aetiology and pathogenesis of scleroderma is incompletely understood. Recently, a cell called the fibrocyte has been shown to be derived from circulating monocytes with the ability to produce collagen. The aim of this study was to evaluate differences in the cell surface characteristics of circulating fibrocyte progenitors (monocytes) in patients with limited scleroderma compared to controls. A case-control study was performed in eight patients with limited scleroderma, which were matched with eight controls. Three-colour flow cytometry was used to assess the relative expression of cell surface markers. Statistical analysis then compared the relative expression between the two groups. In this preliminary study, there were no significant differences in the expression of circulating monocyte surface molecules involved with cell transformation, function, or migration presumed to give rise to fibrocytes, in a population of patients with limited scleroderma. Various explanations for the results are discussed.

  11. Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

    Directory of Open Access Journals (Sweden)

    Phillips Stacia L

    2006-04-01

    Full Text Available Abstract Background We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC. Results Using syngenic mouse embryonic fibroblasts (MEF with wild-type or mutant p53, we now show that, while both cell lines exhibit delays in S/G2 phase post-irradiation, the mutant p53 cells show elevated levels of cyclin B1 followed by MC, while the wild-type p53 cells present both a lower accumulation of cyclin B1 and a lower frequency of MC. Conclusion These results are in line with studies reporting the role of p53 as a post-transcriptional regulator of cyclin B1 protein and confirm that dysregulation of cyclin B1 promote radiation-induced MC. These findings might be exploited to design strategies to augment the yield of MC in tumor cells that are resistant to radiation-induced apoptosis.

  12. Lack of PPARγ in myeloid cells confers resistance to Listeria monocytogenes infection.

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    Zeinab Abdullah

    Full Text Available The peroxisomal proliferator-activated receptor γ (PPARγ is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγ(flox/flox. Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.

  13. Gastro-electric dysrhythm and lack of gastric interstitial cells of cajal

    Institute of Scientific and Technical Information of China (English)

    Qing-Lin Long; Dian-Chun Fang; Hong-Tao Shi; Yuan-Hui Luo

    2004-01-01

    AIM: The pathophysiology underlying gastrointestinal complications of long-standing diabetes is poody understood.Recent evidence suggests an important role of intestitial cells of cajal in controlling gastrointestinal motility. The aim of this study was to clarify the changes of ultrastructural characteristics of interstitial cells of cajal in stomach of diabetic gastro-electric dysrhythmic rats.METHODS: Rats were randomly divided into diabetic group and control group, the model of diabetic rats was established by peritoneally injection of streptozotocin. Electrogastrograms were recorded and intestitial cells of cajal in antrum were observed by electrictelescopy after diabetic model rat was established for 3 mo.RESULTS: In the rats of diabetic group, the gastro-electric dysrhythmia was increased compared with control group,the abnormal rhythm index and the cofficient of variation of slow wave frequency were significantly higher than those of normal rats. The number of the gap junctions of interstitial cells of cajal in antrum of diabetic rats was significantly decreased, and the remaining structures were damaged.The organelles were also damaged, and vacuoles were formed.CONCLUSION: It is possible that changes in ultrastructural characteristics of interstitial cells of cajal in stomach are one of the mechanisms underlying gastro-electric dysrhythm in diabetic rats.

  14. Lack of effect of intranigral transplants of a GABAergic cell line on absence seizures.

    Science.gov (United States)

    Castillo, Claudia G; Mendoza, Soledad; Saavedra, Josie; Giordano, Magda

    2010-08-01

    The substantia nigra pars reticulata (SNpr) is involved in controlling a variety of seizure phenomena. Intranigral transplants of GABAergic cells have been shown to decrease the severity of already established epileptic seizures, but the effects observed have been short-lived. This study evaluated the ability of intranigral transplants of GABA-producing cells to reduce spontaneous absence seizures in a genetic animal model for periods up to 3 months after transplantation. Intranigral transplants did not induce any behavioral deficits in the animals, and they did not form tumors; however, the transplants failed to decrease absence seizures in the genetic model. The assumed increase in intranigral levels of GABA after the transplants may be insufficient to counteract all the factors involved in generating the absence seizures; in this animal model, it may be necessary to further decrease nigral activity by implanting GABAergic cells in another area. These results bear down on the fact that cell transplants need to be tailored for each type of convulsive disorder in terms of the type of cells delivered and the location of the transplants.

  15. Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin.

    Science.gov (United States)

    Haaksma, Carol J; Schwartz, Robert J; Tomasek, James J

    2011-07-01

    Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.

  16. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

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    Carlos Bueno

    Full Text Available Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the

  17. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M.; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  18. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  19. Lack of evidence for superantigen activity of Toxoplasma gondii towards human T cells

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    A.L. Vallochi

    2001-08-01

    Full Text Available Toxoplasma gondii is an obligatory intracellular parasite whose life cycle may include man as an intermediate host. More than 500 million people are infected with this parasite worldwide. It has been previously reported that T. gondii contains a superantigen activity. The purpose of the present study was to determine if the putative superantigen activity of T. gondii would manifest towards human T cells. Peripheral blood mononuclear cells (PBMC from individuals with no previous contact with the parasite were evaluated for proliferation as well as specific Vß expansion after exposure to Toxoplasma antigens. Likewise, PBMC from individuals with the congenital infection were evaluated for putative Vß family deletions in their T cell repertoire. We also evaluated, over a period of one year, the PBMC proliferation pattern in response to Toxoplasma antigens in patients with recently acquired infection. Some degree of proliferation in response to T. gondii was observed in the PBMC from individuals never exposed to the parasite, accompanied by specific Vß expansion, suggesting a superantigen effect. However, we found no specific deletion of Vß (or Valpha families in the blood of congenitally infected individuals. Furthermore, PBMC from recently infected individuals followed up over a period of one year did not present a reduction of the Vß families that were originally expanded in response to the parasite antigens. Taken together, our data suggest that T. gondii does not have a strong superantigen activity on human T cells.

  20. Aberrant neural stem cell proliferation and increased adult neurogenesis in mice lacking chromatin protein HMGB2.

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    Ariel B Abraham

    Full Text Available Neural stem and progenitor cells (NSCs/NPCs are distinct groups of cells found in the mammalian central nervous system (CNS. Previously we determined that members of the High Mobility Group (HMG B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs, suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/- mice exhibit SVZ hyperproliferation, increased numbers of SVZ NSCs, and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM, along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2-/- SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation, and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration.

  1. Enhanced amino acid utilization sustains growth of cells lacking Snf1/AMPK

    DEFF Research Database (Denmark)

    Nicastro, Raffaele; Tripodi, Farida; Guzzi, Cinzia;

    2015-01-01

    The metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregula...

  2. Renal cell carcinoma with rhabdoid-like features lack intracytoplasmic inclusion bodies and show aggressive behavior.

    Science.gov (United States)

    Sugimoto, Masaaki; Kohashi, Kenichi; Kuroiwa, Kentaro; Abe, Tatsuro; Yamada, Yuichi; Shiota, Masaki; Imada, Kenjiro; Naito, Seiji; Oda, Yoshinao

    2016-03-01

    In renal cell carcinoma (RCC), tumor cells with rhabdoid features are characterized by eccentric nuclei, prominent nucleoli, and eosinophilic cytoplasm with intracytoplasmic inclusion bodies. In RCC, tumor cells have also been observed resembling rhabdomyoblasts or rhabdoid but without intracytoplasmic inclusion bodies, and here, we defined these rhabdoid-like features of these cells. To this end, we studied a series of clear cell RCC (ccRCC) with rhabdoid features and compared them with a series of ccRCC with rhabdoid-like features to clarify the differences in the immunohistochemical profile and biological behavior. From 695 cases of ccRCC (80.8 % of all RCCs), 18 cases with rhabdoid features (2.1 % of all RCCs) and 25 cases with rhabdoid-like features (2.9 % of all RCCs) were investigated. The 5-year survival rate for ccRCC with rhabdoid features was 44.7 % and for ccRCC with rhabdoid-like features 30.3 %. Although ccRCC with rhabdoid features showed immunohistochemical co-expression of epithelial markers and vimentin as seen in malignant rhabdoid tumors, ccRCC with rhabdoid-like features showed no such co-expression. Multivariate analyses of cancer-specific survival revealed that perinephric tissues invasion was an independent prognostic factor in ccRCC with rhabdoid features (p = 0.0253) but not in ccRCC with rhabdoid-like features. In summary, although their prognosis is similar, the marker profile and pattern of extension of ccRCC with rhabdoid-like is different from that of ccRCC with rhabdoid features. Therefore, ccRCC with rhabdoid-like features should be distinguished from ccRCC with rhabdoid features.

  3. Delayed contraction of the CD8+ T cell response toward lymphocytic choriomeningitis virus infection in mice lacking serglycin

    DEFF Research Database (Denmark)

    Grujic, Mirjana; Christensen, Jan P; Sørensen, Maria R

    2008-01-01

    We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG...... response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8(+) T cells in SG(-/-) mice. Moreover, wt LCMV-specific CD8(+) T cells from TCR318 transgenic mice...... expanded much more extensively in virus-infected SG(-/-) mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics...

  4. Ataxia and Purkinje cell degeneration in mice lacking the CAMTA1 transcription factor

    Science.gov (United States)

    Long, Chengzu; Grueter, Chad E.; Song, Kunhua; Qin, Song; Qi, Xiaoxia; Kong, Y. Megan; Shelton, John M.; Richardson, James A.; Zhang, Chun-Li; Bassel-Duby, Rhonda; Olson, Eric N.

    2014-01-01

    Members of the calmodulin-binding transcription activator (CAMTA) family of proteins function as calcium-sensitive regulators of gene expression in multicellular organisms ranging from plants to humans. Here, we show that global or nervous system deletion of CAMTA1 in mice causes severe ataxia with Purkinje cell degeneration and cerebellar atrophy, partially resembling the consequences of haploinsufficiency of the human CAMTA1 locus. Gene-expression analysis identified a large collection of neuronal genes that were dysregulated in the brains of CAMTA1-mutant mice, and elucidation of a consensus sequence for binding of CAMTA proteins to DNA revealed the association of CAMTA-binding sites with many of these genes. We conclude that CAMTA1 plays an essential role in the control of Purkinje cell function and survival. CAMTA1-mutant mice provide a model to study the molecular mechanisms of neurodegenerative diseases and for screening potential therapeutic interventions for such disorders. PMID:25049392

  5. Lack of TERT Promoter Mutations in Human B-Cell Non-Hodgkin Lymphoma

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    Gary Lam

    2016-10-01

    Full Text Available Non-Hodgkin lymphomas (NHL are a heterogeneous group of immune cell neoplasms that comprise molecularly distinct lymphoma subtypes. Recent work has identified high frequency promoter point mutations in the telomerase reverse transcriptase (TERT gene of different cancer types, including melanoma, glioma, liver and bladder cancer. TERT promoter mutations appear to correlate with increased TERT expression and telomerase activity in these cancers. In contrast, breast, pancreatic, and prostate cancer rarely demonstrate mutations in this region of the gene. TERT promoter mutation prevalence in NHL has not been thoroughly tested thus far. We screened 105 B-cell lymphoid malignancies encompassing nine NHL subtypes and acute lymphoblastic leukemia, for TERT promoter mutations. Our results suggest that TERT promoter mutations are rare or absent in most NHL. Thus, the classical TERT promoter mutations may not play a major oncogenic role in TERT expression and telomerase activation in NHL.

  6. Lack of TERT Promoter Mutations in Human B-Cell Non-Hodgkin Lymphoma

    Science.gov (United States)

    Lam, Gary; Xian, Rena R.; Li, Yingying; Burns, Kathleen H.; Beemon, Karen L.

    2016-01-01

    Non-Hodgkin lymphomas (NHL) are a heterogeneous group of immune cell neoplasms that comprise molecularly distinct lymphoma subtypes. Recent work has identified high frequency promoter point mutations in the telomerase reverse transcriptase (TERT) gene of different cancer types, including melanoma, glioma, liver and bladder cancer. TERT promoter mutations appear to correlate with increased TERT expression and telomerase activity in these cancers. In contrast, breast, pancreatic, and prostate cancer rarely demonstrate mutations in this region of the gene. TERT promoter mutation prevalence in NHL has not been thoroughly tested thus far. We screened 105 B-cell lymphoid malignancies encompassing nine NHL subtypes and acute lymphoblastic leukemia, for TERT promoter mutations. Our results suggest that TERT promoter mutations are rare or absent in most NHL. Thus, the classical TERT promoter mutations may not play a major oncogenic role in TERT expression and telomerase activation in NHL. PMID:27792139

  7. Mice lacking TrkB in parvalbumin-positive cells exhibit sexually dimorphic behavioral phenotypes

    OpenAIRE

    Lucas, Elizabeth K.; Jegarl, Anita; Clem, Roger L.

    2014-01-01

    Activity-dependent brain-derived neurotrophic factor (BDNF) signaling through receptor tyrosine kinase B (TrkB) is required for cued fear memory consolidation and extinction. Although BDNF is primarily secreted from glutamatergic neurons, TrkB is expressed by other genetically defined cells whose contributions to the behavioral effects of BDNF remain poorly understood. Parvalbumin (PV)-positive interneurons, which are highly enriched in TrkB, are emerging as key regulators of fear memory expr...

  8. Giardiavirus-resistant Giardia lamblia lacks a virus receptor on the cell membrane surface.

    OpenAIRE

    1994-01-01

    Giardia lamblia virus (GLV) is a small nonenveloped double-stranded RNA virus that infects specifically the parasitic protozoan G. lamblia. Among the many collected strains of G. lamblia, a few turn out to be highly resistant to the virus infection. Two of these strains, Ac and JH, were subjected to electroporation with the RNA from GLV-infected G. lamblia WB strain. Subsequent studies indicated the presence of GLV double-stranded RNA and GLV protein in the electroporated and propagated cells...

  9. Mice lacking TrkB in parvalbumin-positive cells exhibit sexually dimorphic behavioral phenotypes.

    Science.gov (United States)

    Lucas, Elizabeth K; Jegarl, Anita; Clem, Roger L

    2014-11-01

    Activity-dependent brain-derived neurotrophic factor (BDNF) signaling through receptor tyrosine kinase B (TrkB) is required for cued fear memory consolidation and extinction. Although BDNF is primarily secreted from glutamatergic neurons, TrkB is expressed by other genetically defined cells whose contributions to the behavioral effects of BDNF remain poorly understood. Parvalbumin (PV)-positive interneurons, which are highly enriched in TrkB, are emerging as key regulators of fear memory expression. We therefore hypothesized that activity-dependent BDNF signaling in PV-interneurons may modulate emotional learning. To test this hypothesis, we utilized the LoxP/Cre system for conditional deletion of TrkB in PV-positive cells to examine the impact of cell-autonomous BDNF signaling on Pavlovian fear conditioning and extinction. However, behavioral abnormalities indicative of vestibular dysfunction precluded the use of homozygous conditional knockouts in tests of higher cognitive functioning. While vestibular dysfunction was apparent in both sexes, female conditional knockouts exhibited an exacerbated phenotype, including extreme motor hyperactivity and circling behavior, compared to their male littermates. Heterozygous conditional knockouts were spared of vestibular dysfunction. While fear memory consolidation was unaffected in heterozygotes of both sexes, males exhibited impaired extinction consolidation compared to their littermate controls. Our findings complement evidence from human and rodent studies suggesting that BDNF signaling promotes consolidation of extinction and point to PV-positive neurons as a discrete population that mediates these effects in a sex-specific manner.

  10. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

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    Ashley A. Zurawel

    2016-09-01

    Full Text Available Proteins containing polyglutamine (polyQ regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD. To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

  11. Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells.

    Science.gov (United States)

    Yamamoto, Daisuke S; Sumitani, Megumi; Kasashima, Katsumi; Sezutsu, Hideki; Matsuoka, Hiroyuki

    2016-09-01

    Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.

  12. Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice.

    Science.gov (United States)

    Skuljec, Jelena; Cabanski, Maciej; Surdziel, Ewa; Lachmann, Nico; Brennig, Sebastian; Pul, Refik; Jirmo, Adan C; Habener, Anika; Visic, Julia; Dalüge, Kathleen; Hennig, Christian; Moritz, Thomas; Happle, Christine; Hansen, Gesine

    2016-07-01

    Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.

  13. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

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    Medler Kathryn F

    2006-03-01

    Full Text Available Abstract Background Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells. Results Depolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells

  14. Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.

    Directory of Open Access Journals (Sweden)

    Minoru Tomizawa

    Full Text Available BACKGROUND: Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction (PCR was performed to analyze the expression of galactokinase 1 (GALK11 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH. The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM, which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E staining, terminal deoxynucleotidyl transferase (TdT-mediated dUTP nick end labeling (TUNEL. The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. RESULTS: The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation, 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. CONCLUSION: Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.

  15. Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

    Science.gov (United States)

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

    2012-01-01

    Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

  16. Studies on responsiveness of hepatoma cells to catecholamines. IV. Lack of adrenergic activation of phosphorylase in rat ascites hepatoma cells.

    Science.gov (United States)

    Miyamoto, K; Yanaoka, T; Sanae, F; Wakusawa, S; Koshiura, R

    1986-10-01

    Glycogen phosphorylase a activity in 7 rat ascites hepatoma cell lines treated with adrenergic agents, phenylephrine, epinephrine and isoproterenol, was investigated as compared with that in freshly isolated rat hepatocytes. Basal phosphorylase activities in hepatoma cells except AH7974 cells were lower than that in hepatocytes. Phosphorylase in hepatoma cells was not activated by any of the agents, while the enzyme activity in hepatocytes was clearly increased in a dose- and time-dependent manner. Phosphorylase in hepatocytes was sensitive to glucagon, but it was found to be insensitive to glucagon in all hepatoma cells. The present results suggest that rat ascites hepatoma cells may escape the glycogenolytic regulation by catecholamines and glucagon.

  17. Mice lacking natural killer T cells are more susceptible to metabolic alterations following high fat diet feeding.

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    Brittany V Martin-Murphy

    Full Text Available Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD. Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d(-/- mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD feeding. Compared with their WT counterparts, CD1d(-/- mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d(-/- mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.

  18. Hydrogen production in a single chamber microbial electrolysis cell lacking a membrane.

    Science.gov (United States)

    Call, Douglas; Logan, Bruce E

    2008-05-01

    Hydrogen gas can be produced by electrohydrogenesis in microbial electrolysis cells (MECs) at greater yields than fermentation and at greater energy efficiencies than water electrolysis. It has been assumed that a membrane is needed in an MEC to avoid hydrogen losses due to bacterial consumption of the product gas. However, high cathodic hydrogen recoveries (78 +/- 1% to 96 +/- 1%) were achieved in an MEC despite the absence of a membrane between the electrodes (applied voltages of 0.3 hydrogen production rates reached a maximum of 3.12 +/- 0.02 m3 H2/m3 reactor per day (292 +/- 1 A/m3) at an applied voltage of E(ap) = 0.8 V. This production rate is more than double that obtained in previous MEC studies. The energy efficiency relative to the electrical input decreased with applied voltage from 406 +/- 6% (E(ap) = 0.3 V) to 194 +/- 2% (E(ap) = 0.8 V). Overall energy efficiency relative to both E(ap) and energy of the substrate averaged 78 +/- 4%, with a maximum of 86 +/- 2% (1.02 +/- 0.05 m3 H2/m3 day, E(ap) = 0.4 V). At E(ap) = 0.2 V, the hydrogen recovery substantially decreased, and methane concentrations increased from an average of 1.9 +/- 1.3% (E(ap) = 0.3-0.8 V) to 28 +/- 0% of the gas, due to the long cycle time of the reactor. Increasing the solution conductivity to 20 mS/ cm increased hydrogen production rates for E(ap) = 0.3-0.6 V, but consistent reactor performance could not be obtained in the high conductivity solution at E(ap) > 0.6 V. These results demonstrate that high hydrogen recovery and production rates are possible in a single chamber MEC without a membrane, potentially reducing the costs of these systems and allowing for new and simpler designs.

  19. Diagnostic utility of hepatocyte nuclear factor 1-beta immunoreactivity in endometrial carcinomas: lack of specificity for endometrial clear cell carcinoma.

    Science.gov (United States)

    Fadare, Oluwole; Liang, Sharon X

    2012-12-01

    cell metaplasia) displayed some degree of HNF1β immunoreactivity, with an average nuclear staining score of 7.3. We conclude that although HNF1β is frequently expressed in clear cell carcinomas, it should be used with caution as a diagnostic marker because of its lack of specificity. It neither distinguishes endometrial serous carcinomas from clear cell carcinomas nor clear cell carcinomas from its benign mimics. The greatest diagnostic utility of HNF1β expression may be in a supportive evidentiary role favoring clear cell carcinoma when the principal differential diagnostic consideration is endometrioid carcinoma.

  20. Lack of PD-L1 expression by iNKT cells improves the course of influenza A infection.

    Directory of Open Access Journals (Sweden)

    Hadi Maazi

    Full Text Available There is evidence indicating that invariant Natural Killer T (iNKT cells play an important role in defense against influenza A virus (IAV. However, the effect of inhibitory receptor, programmed death-1 (PD-1, and its ligands, programmed death ligand (PD-L 1 and 2 on iNKT cells in protection against IAV remains to be elucidated. Here we investigated the effects of these co-stimulatory molecules on iNKT cells in the response to influenza. We discovered that compare to the wild type, PD-L1 deficient mice show reduced sensitivity to IAV infection as evident by reduced weight loss, decreased pulmonary inflammation and cellular infiltration. In contrast, PD-L2 deficient mice showed augmented weight loss, pulmonary inflammation and cellular infiltration compare to the wild type mice after influenza infection. Adoptive transfer of iNKT cells from wild type, PD-L1 or PD-L2 deficient mice into iNKT cell deficient mice recapitulated these findings. Interestingly, in our transfer system PD-L1(-/--derived iNKT cells produced high levels of interferon-gamma whereas PD-L2(-/--derived iNKT cells produced high amounts of interleukin-4 and 13 suggesting a role for these cytokines in sensitivity to influenza. We identified that PD-L1 negatively regulates the frequency of iNKT cell subsets in the lungs of IAV infected mice. Altogether, these results demonstrate that lack of PD-L1 expression by iNKT cells reduces the sensitivity to IAV and that the presence of PD-L2 is important for dampening the deleterious inflammatory responses after IAV infection. Our findings potentially have clinical implications for developing new therapies for influenza.

  1. Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin.

    Directory of Open Access Journals (Sweden)

    Hyoung-Mi Kim

    Full Text Available Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-, are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3 (- transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/- mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/- and Slc26a4(-/- mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/- mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/- mice, and possibly in humans, lacking functional pendrin expression.

  2. Female mice lacking cholecystokinin 1 receptors have compromised neurogenesis, and fewer dopaminergic cells in the olfactory bulb

    Directory of Open Access Journals (Sweden)

    Yi eSui

    2013-03-01

    Full Text Available Neurogenesis in the adult rodent brain is largely restricted to the subependymal zone (SVZ of the lateral ventricle and subgranular zone (SGZ of the dentate gyrus (DG. We examined whether cholecystokinin (CCK through actions mediated by CCK1 receptors (CCK1R is involved in regulating neurogenesis. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 37% and 42%, respectively, in female (but not male mice lacking CCK1Rs (CCK1R-/- compared to wild-type (WT. Generation of neuroblasts in the SVZ and rostral migratory stream was also affected, since the number of doublecortin (DCX-immunoreactive (ir neuroblasts in these regions decreased by 29%. In the SGZ of female CCK1R-/- mice, BrdU-positive (+ and Ki67-ir cells were reduced by 38% and 56%, respectively, while DCX-ir neuroblasts were down 80%. Subsequently, the effect of reduced SVZ/SGZ proliferation on the generation and survival of mature adult-born cells in female CCK1R-/- mice was examined. In the OB granule cell layer (GCL, the number of neuronal nuclei (NeuN-ir and calretinin-ir cells was stable compared to WT, and 42 days after BrdU injections, the number of BrdU+ cells co-expressing GABA- or NeuN-like immunoreactivity (LI was similar. Compared to WT, the granule cell layer of the DG in female CCK1R-/- mice had a similar number of calbindin-ir cells and BrdU+ cells co-expressing calbindin-LI 42 days after BrdU injections. However, the OB glomerular layer (GL of CCK1R-/- female mice had 11% fewer NeuN-ir cells, 23% less TH-ir cells, and a 38% and 29% reduction in BrdU+ cells that co-expressed TH-LI or GABA-LI, respectively. We conclude that CCK, via CCK1Rs, is involved in regulating the generation of proliferating cells and neuroblasts in the adult female mouse brain, and mechanisms are in place to maintain steady neuronal populations in the OB and DG when the rate of proliferation is

  3. Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

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    Yan Mylene L

    2011-08-01

    Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

  4. The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53.

    Science.gov (United States)

    Carney, Bruce K; Caruso Silva, Victoria; Cassimeris, Lynne

    2012-05-01

    In several cancer cell lines, depleting the microtubule (MT)-destabilizing protein stathmin/oncoprotein18 leads to a G2 cell cycle delay and apoptosis. These phenotypes are observed only in synergy with low levels of p53, but the pathway(s) activated by stathmin depletion to delay the cell cycle are unknown. We found that stathmin depletion caused greater MT stability in synergy with loss of p53, measured by the levels of acetylated α-tubulin and the rate of centrosomal MT nucleation. Nocodazole or vinblastine-induced MT depolymerization abrogated the stathmin-depletion induced G2 delay, measured by the percentage of cells staining positive for several markers (TPX2, CDK1 with inhibitory phosphorylation), indicating that MTs are required to lengthen G2. Live cell imaging showed that stathmin depletion increased time in G2 without an impact on the duration of mitosis, indicating that the longer interphase duration is not simply a consequence of a previous slowed mitosis. In contrast, stabilization of MTs with paclitaxel (8 nM) slowed mitosis without lengthening the duration of interphase, demonstrating that increased MT stability alone is not sufficient to delay cells in G2.

  5. Ultrastructural changes in the interstitial cells of Cajal and gastric dysrhythmias in mice lacking full-length dystrophin (mdx mice).

    Science.gov (United States)

    Vannucchi, Maria-Giuliana; Zizzo, Maria-Grazia; Zardo, Claudio; Pieri, Laura; Serio, Rosa; Mulè, Flavia; Faussone-Pellegrini, Maria-Simonetta

    2004-05-01

    At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca(2+) handling, and with defective generation and regeneration of slow wave activity.

  6. Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal

    DEFF Research Database (Denmark)

    Malysz, J; Thuneberg, L; Mikkelsen, Hanne Birte

    1996-01-01

    The small intestine of W/Wv mice lacks both the network of interstitial cells of Cajal (ICC), associated with Auerbach's plexus, and pacemaker activity, i.e., it does not generate slow-wave-type action potentials. The W/Wv muscle preparations showed a wide variety of electrical activities, ranging...... from total quiescence to generation of action potentials at regular or irregular frequency with or without periods of quiescence. The action potentials consisted of a slow component with superimposed spikes, preceded by a slowly developing depolarization and followed by a transient hyperpolarization....... The action potentials were completely abolished by L-type Ca2+ channel blockers. W/Wv mice responded to K+ channel blockade (0.5 mM Ba2+ or 10 mM tetraethylammonium chloride) with effects on amplitude, frequency, rate of rise, and duration of the action potentials. In quiescent tissues from W/Wv mice, K...

  7. Abnormal anxiety- and depression-like behaviors in mice lacking both central serotonergic neurons and pancreatic islet cells.

    Science.gov (United States)

    Jia, Yun-Fang; Song, Ning-Ning; Mao, Rong-Rong; Li, Jin-Nan; Zhang, Qiong; Huang, Ying; Zhang, Lei; Han, Hui-Li; Ding, Yu-Qiang; Xu, Lin

    2014-01-01

    Dysfunction of central serotonin (5-HT) system has been proposed to be one of the underlying mechanisms for anxiety and depression, and the association of diabetes mellitus and psychiatric disorders has been noticed by the high prevalence of anxiety/depression in patients with diabetes mellitus. This promoted us to examine these behaviors in central 5-HT-deficient mice and those also suffering with diabetes mellitus. Mice lacking either 5-HT or central serotonergic neurons were generated by conditional deletion of Tph2 or Lmx1b respectively. Simultaneous depletion of both central serotonergic neurons and pancreatic islet cells was achieved by administration of diphtheria toxin (DT) in Pet1-Cre;Rosa26-DT receptor (DTR) mice. The central 5-HT-deficient mice showed reduced anxiety-like behaviors as they spent more time in and entered more often into the light box in the light/dark box test compared with controls; similar results were observed in the elevated plus maze test. However, they displayed no differences in the immobility time of the forced swimming and tail suspension tests suggesting normal depression-like behaviors in central 5-HT-deficient mice. As expected, DT-treated Pet1-Cre;Rosa26-DTR mice lacking both central serotonergic neurons and pancreatic islet endocrine cells exhibited several classic diabetic symptoms. Interestingly, they displayed increased anxiety-like behaviors but reduced immobility time in the forced swimming and tail suspension tests. Furthermore, the hippocampal neurogenesis was dramatically enhanced in these mice. These results suggest that the deficiency of central 5-HT may not be sufficient to induce anxiety/depression-like behaviors in mice, and the enhanced hippocampal neurogenesis may contribute to the altered depression-like behaviors in the 5-HT-deficient mice with diabetes. Our current investigation provides understanding the relationship between diabetes mellitus and psychiatric disorders.

  8. Abnormal anxiety- and depression-like behaviors in mice lacking both central serotonergic neurons and pancreatic islet cells

    Directory of Open Access Journals (Sweden)

    Yun-Fang eJia

    2014-09-01

    Full Text Available Dysfunction of central serotonin (5-HT system has been proposed to be one of the underlying mechanisms for anxiety and depression, and the association of diabetes mellitus and psychiatric disorders has been noticed by the high prevalence of anxiety/depression in patients with diabetes mellitus. This promoted us to examine these behaviors in central 5-HT-deficient mice and those also suffering with diabetes mellitus. Mice lacking either 5-HT or central serotonergic neurons were generated by conditional deletion of Tph2 or Lmx1b respectively. Simultaneous depletion of both central serotonergic neurons and pancreatic islet cells was achieved by administration of diphtheria toxin (DT in Pet1-Cre;Rosa26-DT receptor (DTR mice. The central 5-HT-deficient mice showed reduced anxiety-like behaviors as they spent more time in and entered more often into the light box in the light/dark box test compared with controls; similar results were observed in the elevated plus maze test. However, they displayed no differences in the immobility time of the forced swimming and tail suspension tests suggesting normal depression-like behaviors in central 5-HT-deficient mice. As expected, DT-treated Pet1-Cre;Rosa26-DTR mice lacking both central serotonergic neurons and pancreatic islet endocrine cells exhibited several classic diabetic symptoms. Interestingly, they displayed increased anxiety-like behaviors but reduced immobility time in the forced swimming and tail suspension tests. Furthermore, the hippocampal neurogenesis was dramatically enhanced in these mice. These results suggest that the deficiency of central 5-HT may not be sufficient to induce anxiety/depression-like behaviors in mice, and the enhanced hippocampal neurogenesis may contribute to the altered depression-like behaviors in the 5-HT-deficient mice with diabetes. Our current investigation provides a novel insight into understanding the relationship between diabetes mellitus and psychiatric disorders.

  9. Lack of liver X receptors leads to cell proliferation in a model of mouse dorsal prostate epithelial cell.

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    Julie Dufour

    Full Text Available Recent studies underline the implication of Liver X Receptors (LXRs in several prostate diseases such as benign prostatic hyperplasia (BPH and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs of wild type (WT or Lxrαβ-/- mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ-/- mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer.

  10. The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

    DEFF Research Database (Denmark)

    Baek, Kristoffer T.; Bowman, Lisa; Millership, Charlotte;

    2016-01-01

    Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise...

  11. The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

    DEFF Research Database (Denmark)

    Baek, Kristoffer T.; Bowman, Lisa; Millership, Charlotte;

    2016-01-01

    Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise ...

  12. Stathmin/oncoprotein 18, a microtubule regulatory protein, is required for survival of both normal and cancer cell lines lacking the tumor suppressor, p53.

    Science.gov (United States)

    Carney, Bruce K; Cassimeris, Lynne

    2010-05-01

    Stathmin, a microtubule regulatory protein, is overexpressed in many cancers and required for survival of several cancer lines. In a study of breast cancer cell lines(1) proposed that stathmin is required for survival of cells lacking p53, but this hypothesis was not tested directly. Here we tested their hypothesis by examining cell survival in cells depleted of stathmin, p53 or both proteins. Comparing HCT116 colon cancer cell lines differing in TP53 genotype, stathmin depletion resulted in significant death only in cells lacking p53. As a second experimental system, we compared the effects of stathmin depletion from HeLa cells, which normally lack detectable levels of p53 due to expression of the HPV E6 protein. Stathmin depletion caused a large percentage of HeLa cells to die. Restoring p53, by depletion of HPV E6, rescued HeLa cells from stathmin-depletion induced death. Cleaved PARP was detected in HCT116(p53-/-) cells depleted of stathmin and cell death in stathmin-depleted HeLa cells was blocked by the caspase inhibitor Z-VAD-FMK, consistent with apoptotic death. The stathmin-dependent survival of cells lacking p53 was not confined to cancerous cells because both proteins were required for survival of normal human fibroblasts. In HCT116 and HeLa cells, depletion of both stathmin and p53 leads to a cell cycle delay through G(2). Our results demonstrate that stathmin is required for cell survival in cells lacking p53, suggesting that stathmin depletion could be used therapeutically to induce apoptosis in tumors without functional p53.

  13. Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

    OpenAIRE

    Ribeiro,Daniel Araki; Alves de Lima, Patrícia Lepage; Marques, Mariângela Esther Alencar; Salvadori,Daisy Maria Favero

    2006-01-01

    Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and hum...

  14. Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

    Science.gov (United States)

    El-Khamisy, Sherif F.; Katyal, Sachin; Patel, Poorvi; Ju, Limei; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1. PMID:19303373

  15. Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.

    Science.gov (United States)

    Tiemessen, Machteld M; Mitchell, Tracey J; Hendry, Lisa; Whittaker, Sean J; Taams, Leonie S; John, Susan

    2006-10-01

    Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

  16. Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

    Science.gov (United States)

    Uciechowski, Peter; Imhoff, Heidi; Lange, Christoph; Meyer, Christian G; Browne, Edmund N; Kirsten, Detlef K; Schröder, Anja K; Schaaf, Bernhard; Al-Lahham, Adnan; Reinert, Ralf René; Reiling, Norbert; Haase, Hajo; Hatzmann, Adelheid; Fleischer, Daniela; Heussen, Nicole; Kleines, Michael; Rink, Lothar

    2011-08-01

    Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real-time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU-C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU-C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low-incidence country.

  17. An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormone-dependent cancer cells.

    Science.gov (United States)

    Shields-Botella, J; Duc, I; Duranti, E; Puccio, F; Bonnet, P; Delansorne, R; Paris, J

    2003-11-01

    The specific pharmacological profile of the 19-norprogestin nomegestrol acetate (NOMAC) is, at least in part, defined by its pattern of binding affinities to the different steroid hormone receptors. In the present study, its affinity to the progesterone receptor (PgR), the androgen receptor (AR) and the estrogen receptor (ER) was re-evaluated and compared to those obtained for progesterone (P) and several progestins. The characteristics of binding to the PgR in rat uterus were determined and Ki were found to be roughly similar with 22.8 and 34.3 nM for NOMAC and P, respectively. The binding characteristics of 3H-NOMAC were also determined and compared to that of 3H-ORG2058 with Kd of 5 and 0.6 nM, respectively for rat uterus and 4 and 3 nM, respectively for human T47-D cells. Structure-affinity and -activity relationships were studied on a variety of compounds related to NOMAC in order to assess its specificity as a progestin. The effects of NOMAC on the binding of androgen to the AR were investigated, using rat ventral prostate as target model. Contrary to what was observed for MPA, the RBA of NOMAC was found to decline with time, showing anti-androgenic rather than androgenic potential, a result that was confirmed in vivo. Regarding the ER, since none of the progestins were able to compete with estrogen for binding in rat uterus as well as in Ishikawa cells, the induction of alkaline phosphatase activity (APase) was used as an estrogen-specific response. It confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT), norethisterone acetate (NETA), levonorgestrel (LNG) or norgestimate (NGM) and others. In contrast, all P and 19-norP derivatives remained inactive. Finally, to complete this overview of NOMAC at the sex steroid receptor levels, the lack of estrogenic or estrogenic-like activity was checked out in different in vitro models. Data from this study have demonstrated that NOMAC is a progestin that has greater steroid

  18. Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death.

    Science.gov (United States)

    Robson, Christine A; Vanlerberghe, Greg C

    2002-08-01

    The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.

  19. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.

    Science.gov (United States)

    Kawabe, Keiko; Tateyama, Daiki; Toyoda, Hidenao; Kawasaki, Nana; Hashii, Noritaka; Nakao, Hiromi; Matsumoto, Shogo; Nonaka, Motohiro; Matsumura, Hiroko; Hirose, Yoshinori; Morita, Ayaha; Katayama, Madoka; Sakuma, Makoto; Kawasaki, Nobuko; Furue, Miho Kusuda; Kawasaki, Toshisuke

    2013-03-01

    We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

  20. Innate Response to Human Cancer Cells with or without IL-2 Receptor Common γ-Chain Function in NOD Background Mice Lacking Adaptive Immunity.

    Science.gov (United States)

    Nishime, Chiyoko; Kawai, Kenji; Yamamoto, Takehiro; Katano, Ikumi; Monnai, Makoto; Goda, Nobuhito; Mizushima, Tomoko; Suemizu, Hiroshi; Nakamura, Masato; Murata, Mitsuru; Suematsu, Makoto; Wakui, Masatoshi

    2015-08-15

    Immunodeficient hosts exhibit high acceptance of xenogeneic or neoplastic cells mainly due to lack of adaptive immunity, although it still remains to be elucidated how innate response affects the engraftment. IL-2R common γ-chain (IL-2Rγc) signaling is required for development of NK cells and a subset of dendritic cells producing IFN-γ. To better understand innate response in the absence of adaptive immunity, we examined amounts of metastatic foci in the livers after intrasplenic transfer of human colon cancer HCT116 cells into NOD/SCID versus NOD/SCID/IL-2Rγc (null) (NOG) hosts. The intravital microscopic imaging of livers in the hosts depleted of NK cells and/or macrophages revealed that IL-2Rγc function critically contributes to elimination of cancer cells without the need for NK cells and macrophages. In the absence of IL-2Rγc, macrophages play a role in the defense against tumors despite the NOD Sirpa allele, which allows human CD47 to bind to the encoded signal regulatory protein α to inhibit macrophage phagocytosis of human cells. Analogous experiments using human pancreas cancer MIA PaCa-2 cells provided findings roughly similar to those from the experiments using HCT116 cells except for lack of suppression of metastases by macrophages in NOG hosts. Administration of mouse IFN-γ to NOG hosts appeared to partially compensate lack of IL-2Rγc-dependent elimination of transferred HCT116 cells. These results provide insights into the nature of innate response in the absence of adaptive immunity, aiding in developing tumor xenograft models in experimental oncology.

  1. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction.

    Science.gov (United States)

    Lodie, Tracey A; Blickarz, Courtney E; Devarakonda, Tara J; He, Chufa; Dash, Ajeeta B; Clarke, Jennifer; Gleneck, Kristen; Shihabuddin, Lamya; Tubo, Ross

    2002-10-01

    Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

  2. LACK OF INHIBITORY ACTION OF OXYTOCIN,VASOTOCIN AND VASOPRESSIN ON PROGESTERONE PRODUCTION BY HUMAN LUTEAL CELLS

    Institute of Scientific and Technical Information of China (English)

    WANGHan-Zheng; HANXiang-Jun; LUShu-Hua; SUNZhi-Da; SHENWei-Xiong; ZHOUWei

    1989-01-01

    The direct effects of oxytocin and relatcd peptides on human luteal cells were attudied Suslxmsions of luteal cells were prepared enzymatically from human corpora htae, obtained during cycle days 19-25. Afar incubation with hCG (liu / ml) for 3 h, the

  3. Lack of cardiac differentiation in c-kit-enriched porcine bone marrow and spleen hematopoietic cell cultures using 5-azacytidine

    NARCIS (Netherlands)

    M.L. Ramirez (Mario); T. McMorrow (Tara); T.M. Sanderson (Thomas M.); C.J. Lancos (C.); Y.-L. Tseng (Y.); D.K.C. Cooper (David); F.J.M.F. Dor (Frank)

    2005-01-01

    textabstractThe adult spleen is a source of early hematopoietic stem cells (HSC). We therefore studied whether culturing spleen or bone marrow (BM) HSC in medium containing 5-azacytidine could induce a cardiac phenotype. c-kit enrichment and depletion of adult pig spleen and BM mononuclear cells wer

  4. "The preadipocyte factor" DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Jensen, Line; Schrøder, Henrik Daa;

    2009-01-01

    Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as "the" preadipocyte marker, yet the phenotype of DLK1(+) cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1(+) cells encompass around 1-2% of the adult mouse adipose stromal...

  5. Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain...... of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell...... membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. Methods: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3...

  6. Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling.

    Science.gov (United States)

    Park, Tae Joo; Haigo, Saori L; Wallingford, John B

    2006-03-01

    The vertebrate planar cell polarity (PCP) pathway has previously been found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP effectors inturned (in) or fuzzy (fy) affected not only PCP-dependent convergent extension but also elicited embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins govern apical actin assembly and thus control the orientation, but not assembly, of ciliary microtubules. Finally, accumulation of Dishevelled and Inturned near the basal apparatus of cilia suggests that these proteins function in a common pathway with core PCP components to regulate ciliogenesis. Together, these data highlight the interrelationships between cell polarity, cellular morphogenesis, signal transduction and cell fate specification.

  7. Co-existence of intact stemness and priming of neural differentiation programs in mES cells lacking Trim71

    Science.gov (United States)

    Mitschka, Sibylle; Ulas, Thomas; Goller, Tobias; Schneider, Karin; Egert, Angela; Mertens, Jérôme; Brüstle, Oliver; Schorle, Hubert; Beyer, Marc; Klee, Kathrin; Xue, Jia; Günther, Patrick; Bassler, Kevin; Schultze, Joachim L.; Kolanus, Waldemar

    2015-01-01

    Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stemness and differentiation genes. Translational regulators such as RNA binding proteins (RBPs) and micro RNAs (miRNAs) might be prime candidates for guiding a cell from pluripotency to differentiation. Using Trim71, one of two members of the Tripartite motif (Trim) protein family with RNA binding activity expressed in murine ES cells, we demonstrate that Trim71 is not involved in regulatory networks of pluripotency but regulates neural differentiation. Loss of Trim71 in mES cells leaves stemness and self-maintenance of these cells intact, but many genes required for neural development are up-regulated at the same time. Concordantly, Trim71−/− mES show increased neural marker expression following treatment with retinoic acid. Our findings strongly suggest that Trim71 keeps priming steps of differentiation in check, which do not pre-require a loss of the pluripotency network in ES cells. PMID:26057209

  8. Co-existence of intact stemness and priming of neural differentiation programs in mES cells lacking Trim71.

    Science.gov (United States)

    Mitschka, Sibylle; Ulas, Thomas; Goller, Tobias; Schneider, Karin; Egert, Angela; Mertens, Jérôme; Brüstle, Oliver; Schorle, Hubert; Beyer, Marc; Klee, Kathrin; Xue, Jia; Günther, Patrick; Bassler, Kevin; Schultze, Joachim L; Kolanus, Waldemar

    2015-06-09

    Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stemness and differentiation genes. Translational regulators such as RNA binding proteins (RBPs) and micro RNAs (miRNAs) might be prime candidates for guiding a cell from pluripotency to differentiation. Using Trim71, one of two members of the Tripartite motif (Trim) protein family with RNA binding activity expressed in murine ES cells, we demonstrate that Trim71 is not involved in regulatory networks of pluripotency but regulates neural differentiation. Loss of Trim71 in mES cells leaves stemness and self-maintenance of these cells intact, but many genes required for neural development are up-regulated at the same time. Concordantly, Trim71(-/-) mES show increased neural marker expression following treatment with retinoic acid. Our findings strongly suggest that Trim71 keeps priming steps of differentiation in check, which do not pre-require a loss of the pluripotency network in ES cells.

  9. Motor dysfunction and altered synaptic transmission at the parallel fiber-Purkinje cell synapse in mice lacking potassium channels Kv3.1 and Kv3.3.

    Science.gov (United States)

    Matsukawa, Hiroshi; Wolf, Alexander M; Matsushita, Shinichi; Joho, Rolf H; Knöpfel, Thomas

    2003-08-20

    Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.

  10. Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Kauffmann, Susanne Ørding; Thomsen, Allan Randrup

    2003-01-01

    precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B(-/-) mice to produce neutralizing Abs. In contrast with CD8(+) T cells, which initially respond almost as in wild-type mice, the priming...

  11. Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

    DEFF Research Database (Denmark)

    Breau, Marie A; Pietri, Thomas; Eder, Olivier

    2006-01-01

    crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover, beta1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network...... organisation. Organotypic cultures of gut explants reveal that beta1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that beta1...

  12. Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM.

    Science.gov (United States)

    So, Sairei; Adachi, Noritaka; Koyama, Hideki

    2007-07-01

    The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(/)TP53(/) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.

  13. Co-existence of intact stemness and priming of neural differentiation programs in mES cells lacking Trim71

    OpenAIRE

    Mitschka, Sibylle; Ulas, Thomas; Goller, Tobias; Schneider, Karin; Egert, Angela; Mertens, Jérôme; Brüstle, Oliver; Schorle, Hubert; Beyer, Marc; Klee, Kathrin; Xue, Jia; Günther, Patrick; Bassler, Kevin; Schultze, Joachim L; Kolanus, Waldemar

    2015-01-01

    Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stem...

  14. Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV.

    Science.gov (United States)

    Diaz, Francisca; Enríquez, José Antonio; Moraes, Carlos T

    2012-01-01

    Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O(2)) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.

  15. T cell clones from Schistosoma haematobium infected and exposed individuals lacking distinct cytokine profiles for Th1/Th2 polarisation

    Directory of Open Access Journals (Sweden)

    Mduluza T

    2001-01-01

    Full Text Available T cell clones were derived from peripheral blood mononuclear cells of Schistosoma haematobium infected and uninfected individuals living in an endemic area. The clones were stimulated with S. haematobium worm and egg antigens and purified protein derivative. Attempts were made to classify the T cell clones according to production of the cytokines IL-4, IL-5 and IFN-gamma. All the T cell clones derived were observed to produce cytokines used as markers for the classification of Th1/Th2 subsets. However, the 'signature' cytokines marking each subset were produced at different levels. The classification depended on the dominating cytokine type, which was having either Th0/1 or Th0/2 subsets. The results indicated that no distinct cytokine profiles for polarisation of Th1/Th2 subsets were detected in these S. haematobium infected humans. The balance in the profiles of cytokines marking each subset were related to infection and re-infection status after treatment with praziquantel. In the present study, as judged by the changes in infection status with time, the T cell responses appeared to be less stable and more dynamic, suggesting that small quantitative changes in the balance of the cytokines response could result in either susceptibility or resistant to S. haematobium infection.

  16. Hypocholesterolemia, foam cell accumulation, but no atherosclerosis in mice lacking ABC-transporter A1 and scavenger receptor BI

    NARCIS (Netherlands)

    Zhao, Ying; Pennings, Marieke; Vrins, Carlos L. J.; Calpe-Berdiel, Laura; Hoekstra, Menno; Kruijt, J. Kar; Ottenhoff, Roelof; Hildebrand, Reeni B.; van der Sluis, Ronald; Jessup, Wendy; Le Goff, Wilfried; Chapman, M. John; Huby, Thierry; Groen, Albert K.; Van Berkel, Theo J. C.; Van Eck, Miranda

    2011-01-01

    High-density lipoprotein (HDL) mediated reverse cholesterol transport (RCT) is regarded to be crucial for prevention of foam cell formation and atherosclerosis. ABC-transporter A1 (ABCA1) and scavenger receptor BI (SR-BI) are involved in the biogenesis of HDL and the selective delivery of HDL choles

  17. Lack of CCR5 on dendritic cells promotes a proinflammatory environment in submandibular glands of the NOD mouse

    NARCIS (Netherlands)

    M.E. Wildenberg; C.G. van Helden-Meeuwsen; J.P. van de Merwe (Joop); C. Moreno (Christophe); H.A. Drexhage (Hemmo); M.A. Versnel (Marjan)

    2008-01-01

    textabstractSjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltration of the salivary glands. In the NOD mouse, a model for this disease, the development of lymphocytic infiltrates in the salivary glands is preceded by an accumulation of dendritic cells (DC). Given the ke

  18. Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells.

    Science.gov (United States)

    Rauner, Martina; Franke, Kristin; Murray, Marta; Singh, Rashim Pal; Hiram-Bab, Sahar; Platzbecker, Uwe; Gassmann, Max; Socolovsky, Merav; Neumann, Drorit; Gabet, Yankel; Chavakis, Triantafyllos; Hofbauer, Lorenz C; Wielockx, Ben

    2016-10-01

    The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2(f/f) ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2(f/f) ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2(f/f) ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

  19. Lowering GTP level increases survival of amino acid starvation but slows growth rate for Bacillus subtilis cells lacking (p)ppGpp.

    Science.gov (United States)

    Bittner, Alycia N; Kriel, Allison; Wang, Jue D

    2014-06-01

    Bacterial cells sense external nutrient availability to regulate macromolecular synthesis and consequently their growth. In the Gram-positive bacterium Bacillus subtilis, the starvation-inducible nucleotide (p)ppGpp negatively regulates GTP levels, both to resist nutritional stress and to maintain GTP homeostasis during growth. Here, we quantitatively investigated the relationship between GTP level, survival of amino acid starvation, and growth rate when GTP synthesis is uncoupled from its major homeostatic regulator, (p)ppGpp. We analyzed growth and nucleotide levels in cells that lack (p)ppGpp and found that their survival of treatment with a nonfunctional amino acid analog negatively correlates with both growth rate and GTP level. Manipulation of GTP levels modulates the exponential growth rate of these cells in a positive dose-dependent manner, such that increasing the GTP level increases growth rate. However, accumulation of GTP levels above a threshold inhibits growth, suggesting a toxic effect. Strikingly, adenine counteracts GTP stress by preventing GTP accumulation in cells lacking (p)ppGpp. Our results emphasize the importance of maintaining appropriate levels of GTP to maximize growth: cells can survive amino acid starvation by decreasing GTP level, which comes at a cost to growth, while (p)ppGpp enables rapid adjustment to nutritional stress by adjusting GTP level, thus maximizing fitness.

  20. Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation.

    Science.gov (United States)

    Mortensen, Monika; Watson, Alexander Scarth; Simon, Anna Katharina

    2011-09-01

    The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).

  1. Lack of relationship between TIMP-1 tumour cell immunoreactivity, treatment efficacy and prognosis in patients with advanced epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Christensen, Rikke Kølby

    2010-01-01

    BACKGROUND: Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a natural inhibitor of the matrix metalloproteinases (MMPs) which are proteolytic enzymes involved in degradation of extracellular matrix thereby favoring tumour cell invasion and metastasis. TIMP-1 activity in tumour tissue may ther...... immunoreactivity in tumour tissue from patients with primary epithelial ovarian cancer did not correlate with patient survival or response to combination platinum/cyclophosphamide therapy.......BACKGROUND: Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a natural inhibitor of the matrix metalloproteinases (MMPs) which are proteolytic enzymes involved in degradation of extracellular matrix thereby favoring tumour cell invasion and metastasis. TIMP-1 activity in tumour tissue may...... therefore play an essential role in the progression of a malignant tumour.The primary aim of the present study was to evaluate TIMP-1 protein immunoreactivity in tissue from primary ovarian cancer patients and associate these findings with the course of the disease including response to treatment...

  2. Mast cells are responsible for the lack of anti-inflammatory effects of morphine in CBA mice

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    Elzbieta Stankiewicz

    2004-01-01

    Full Text Available BACKGROUND and aim: Morphine co-injection has anti-inflammatory effects on zymosan-induced peritonitis in several strains of mice except that of CBA. As peritoneal mast cells (pMCs are much more numerous in CBA mice than in SWISS mice, the role of pMCs in morphine-modulated zymosan peritonitis is compared in CBA and SWISS males.

  3. Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells.

    Science.gov (United States)

    Felmy, Felix

    2009-06-01

    Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-beta-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-beta-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal-spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

  4. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

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    Sowa, Marianne B.; Goetz, Wilfried; Baulch, Janet E.; Pyles, Dinah N.; Dziegielewski, J.; Yovino, Susannah; Snyder, Andrew R.; de Toledo, S. M.; Azzam, Edouard I.; Morgan, William F.

    2010-02-01

    The conventional paradigm in radiation biology has been that DNA is the primary target for energy deposition following exposure to ionizing radiation. However, studies focusing on the non-target effects of radiation, i.e. effects occurring in cells not directly exposed to radiation, imply that the target of exposure is larger than what has traditionally been assumed and could have significant implications for radiation health risks. We have conducted an extensive study of the low-LET bystander effect including multiple cell lines and endpoints and various radiation sources and exposure scenarios. In no instance do we see evidence of a low-LET induced bystander effect. However, direct comparison for alpha particle exposure showed a statistically significant media transfer bystander effect for high-LET but not for low-LET radiation. From our results it is evident that there are many confounding factors mitigating bystander responses as reported in the literature and for the cell lines we studied that there is a LET dependence for the observed responses. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

  5. Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark.

    Science.gov (United States)

    Chaudhari, Vishalsingh R; Vyawahare, Aniket; Bhattacharjee, Swapan K; Rao, Basuthkar J

    2015-03-01

    Plant cells are known to differentiate their responses to stress depending up on the light conditions. We observed that UVC sensitive phenotype of light grown asynchronous Chlamydomonas reinhardtii culture (Light culture: LC) can be converted to relatively resistant form by transfer to dark condition (Dark culture: DC) before UVC exposure. The absence of photosystem II (PSII) function, by either atrazine treatment in wild type or in D1 (psbA) null mutant, conferred UV protection even in LC. We provide an indirect support for involvement of reactive oxygen species (ROS) signalling by showing higher UV survival on exposures to mild dose of H2O2 or Methyl Viologen. Circadian trained culture also showed a rhythmic variation in UV sensitivity in response to alternating light-dark (12 h:12 h) entrainment, with maximum UV survival at the end of 12 h dark and minimum at the end of 12 h light. This rhythm failed to maintain in "free running" conditions, making it a non-circadian phenotype. Moreover, atrazine strongly inhibited rhythmic UV sensitivity and conferred a constitutively high resistance, without affecting internal circadian rhythm marker expression. Dampening of UV sensitivity rhythm in Thymine-dimer excision repair mutant (cc-888) suggested the involvement of DNA repair in this phenomenon. DNA excision repair (ER) assays in cell-free extracts revealed that dark incubated cells exhibit higher ER compared to those growing in light, underscoring the role of ER in conferring differential UV sensitivity in dark versus light incubation. We suggest that multiple factors such as ROS changes triggered by differences in PSII activity, concomitant with differential ER efficiency collectively contribute to light-dark (12 h: 12 h) rhythmicity in C. reinhardtii UV sensitivity.

  6. A form of mitofusin 2 (Mfn2) lacking the transmembrane domains and the COOH-terminal end stimulates metabolism in muscle and liver cells.

    Science.gov (United States)

    Segalés, Jessica; Paz, José C; Hernández-Alvarez, María Isabel; Sala, David; Muñoz, Juan Pablo; Noguera, Eduard; Pich, Sara; Palacín, Manuel; Enríquez, José Antonio; Zorzano, Antonio

    2013-11-15

    Mitofusin 2 (Mfn2), a protein that participates in mitochondrial fusion, is required to maintain normal mitochondrial metabolism in skeletal muscle and liver. Given that muscle Mfn2 is repressed in obese or type 2 diabetic subjects, this protein may have a potential pathophysiological role in these conditions. To evaluate whether the metabolic effects of Mfn2 can be dissociated from its function in mitochondrial dynamics, we studied a form of human Mfn2, lacking the two transmembrane domains and the COOH-terminal coiled coil (ΔMfn2). This form localized in mitochondria but did not alter mitochondrial morphology in cells or in skeletal muscle fibers. The expression of ΔMfn2 in mouse skeletal muscle stimulated glucose oxidation and enhanced respiratory control ratio, which occurred in the absence of changes in mitochondrial mass. ΔMfn2 did not stimulate mitochondrial respiration in Mfn2-deficient muscle cells. The expression of ΔMfn2 in mouse liver or in hepatoma cells stimulated gluconeogenesis. In addition, ΔMfn2 activated basal and maximal respiration both in muscle and liver cells. In all, we show that a form of Mfn2 lacking mitochondrial fusion activity stimulates mitochondrial function and enhances glucose metabolism in muscle and liver tissues. This study suggests that Mfn2 regulates metabolism independently of changes in mitochondrial morphology.

  7. Efficient CRISPR-Cas9-Mediated Generation of Knockin Human Pluripotent Stem Cells Lacking Undesired Mutations at the Targeted Locus

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    Florian T. Merkle

    2015-05-01

    Full Text Available The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs, but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

  8. Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis.

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    Xiaobo Liang

    Full Text Available BACKGROUND: COUP-TF interacting protein 2 [(Ctip2, also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2(ep-/- mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB establishment during development, similar to what was observed in Ctip2 null (Ctip2(-/- mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing. METHODOLOGY/PRINCIPAL FINDINGS: Full thickness excisional wound healing experiments were performed on Ctip2(L2/L2 and Ctip2(ep-/- animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2(ep-/- mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair. CONCLUSIONS/SIGNIFICANCE: Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure.

  9. Human leukocyte antigen-G expression in differentiated human airway epithelial cells: lack of modulation by Th2-associated cytokines

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    White Steven R

    2013-01-01

    Full Text Available Abstract Background Human leukocyte antigen (HLA-G is a nonclassical class I antigen with immunomodulatory roles including up-regulation of suppressor T regulatory lymphocytes. HLA-G was recently identified as an asthma susceptibility gene, and expression of a soluble isoform, HLA-G5, has been demonstrated in human airway epithelium. Increased presence of HLA-G5 has been demonstrated in bronchoalveolar lavage fluid recovered from patients with mild asthma; this suggests a role for this isoform in modulating airway inflammation though the mechanisms by which this occurs is unclear. Airway inflammation associated with Th2 cytokines such as IL-4 and IL-13 is a principal feature of asthma, but whether these cytokines elicit expression of HLA-G is not known. Methods We examined gene and protein expression of both soluble (G5 and membrane-bound (G1 HLA-G isoforms in primary differentiated human airway epithelial cells collected from normal lungs and grown in air-liquid interface culture. Cells were treated with up to 10 ng/ml of either IL-4, IL-5, or IL-13, or 100 ng/ml of the immunomodulatory cytokine IL-10, or 10,000 U/ml of the Th1-associated cytokine interferon-beta, for 24 hr, after which RNA was isolated for evaluation by quantitative PCR and protein was collected for Western blot analysis. Results HLA-G5 but not G1 was present in dAEC as demonstrated by quantitative PCR, western blot and confocal microscopy. Neither G5 nor G1 expression was increased by the Th2-associated cytokines IL-4, IL-5 or IL-13 over 24 hr, nor after treatment with IL-10, but was increased 4.5 ± 1.4 fold after treatment with 10,000 U/ml interferon-beta. Conclusions These data demonstrate the constitutive expression of a T lymphocyte regulatory molecule in differentiated human airway epithelial cells that is not modulated by Th2-associated cytokines.

  10. Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model.

    Science.gov (United States)

    Dusabineza, Ange-Clarisse; Van Hul, Noémi K; Abarca-Quinones, Jorge; Starkel, Peter; Najimi, Mustapha; Leclercq, Isabelle A

    2012-01-01

    When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ∼100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, PPH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (Plivers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.

  11. Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ashton, Thomas M; Hickson, Ian D

    2011-01-01

    The Sgs1-Rmi1-Top3 "dissolvasome" is required for the maintenance of genome stability and has been implicated in the processing of various types of DNA structures arising during DNA replication. Previous investigations have revealed that unprocessed (X-shaped) homologous recombination repair (HRR......) intermediates persist when S-phase is perturbed by using methyl methanesulfonate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 or Top3. However, the precise nature of these persistent DNA structures remains poorly characterized. Here, we report that ectopic expression of either of two heterologous...... and structurally unrelated Holliday junction (HJ) resolvases, Escherichia coli RusA or human GEN1(1-527), promotes the removal of these X-structures in vivo. Moreover, other types of DNA replication intermediates, including stalled replication forks and non-HRR-dependent X-structures, are refractory to RusA or GEN...

  12. Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma.

    Science.gov (United States)

    Pedersen, Mette Ø; Poulsen, Tim S; Gang, Anne O; Knudsen, Helle; Lauritzen, Anne F; Pedersen, Michael; Nielsen, Signe L; Brown, Peter; Høgdall, Estrid; Nørgaard, Peter

    2015-07-01

    Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number changes at relapse, with possible implications for relapse therapy efficacy. TOP gene alterations in lymphoma are poorly investigated. In this study, TOP1 and TOP2A gene alterations were investigated in patients with de novo diffuse large B-cell lymphoma (DLBCL) (n = 33) and relapsed DLBCL treated with chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13 patients (38%) and 4 of 16 patients (25%) harboring chromosome 20 and 17 polysomy, respectively; however, these differences only tended to be significant (p = 0.09 and p = 0.09, respectively). The results suggest that TOP gene copy number changes are very infrequent in DLBCL and not likely induced by TOP2-targeting drugs. Increased polyploidy of chromosomes 17 and 20 among patients with relapsed DLBCL may reflect genetic compensation in the tumor cells after TOP2 inhibition, but is more likely due to the increased genetic instability often seen in progressed cancers. Therefore, it is unlikely that TOP1 and TOP2A gene alterations can be used as predictive markers for response to treatment with TOP2-targeting drugs in patients with DLBCL.

  13. CD8 T-cells from most HIV-infected patients lack ex vivo HIV-suppressive capacity during acute and early infection.

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    Camille Lécuroux

    Full Text Available The strong CD8+ T-cell-mediated HIV-1-suppressive capacity found in a minority of HIV-infected patients in chronic infection is associated with spontaneous control of viremia. However, it is still unclear whether such capacities were also present earlier in the CD8+ T cells from non controller patients and then lost as a consequence of uncontrolled viral replication. We studied 50 patients with primary HIV-1-infection to determine whether strong CD8+ T-cell-mediated HIV suppression is more often observed at that time. Despite high frequencies of polyfunctional HIV-specific CD8+ T-cells and a strong CD4+ T-helper response, CD8+ T-cells from 48 patients lacked strong HIV-suppressive capacities ex vivo. This indicates that the superior HIV-suppressive capacity of CD8+ T-cells from HIV controllers is not a general characteristic of the HIV-specific CD8+ T cell response in primary HIV infection.

  14. Contribution of Nrf2 to Atherogenic Phenotype Switching of Coronary Arterial Smooth Muscle Cells Lacking CD38 Gene

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    Ming Xu

    2015-08-01

    Full Text Available Background/Aims: Recent studies have indicated that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle cells upon atherogenic stimulations. However, the molecular mechanisms mediating this vascular smooth muscle (SMC phenotypic switching remain unknown. Methods & Results: In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs from CD38-/- mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin, SM22α and α-SMA but increased the expression of SMC dedifferentiation marker, vimentin, which was accompanied by enhanced cell proliferation. This phenotypic change in CD38-/- CAMs was enhanced by 7-ketocholesterol (7-Ket, an atherogenic stimulus. We further found that the CD38 deficiency decreased the expression and activity of nuclear factor E2-related factor 2 (Nrf2, a basic leucine zipper (bZIP transcription factor sensitive to redox regulation. Similar to CD38 deletion, Nrf2 gene silencing increased CAM dedifferentiation upon 7-Ket stimulation. In contrast, the overexpression of Nrf2 gene abolished 7-Ket-induced dedifferentiation in CD38-/- CAMs. Given the sensitivity of Nrf2 to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity associated with CD38 effect in CAM phenotype changes. It was demonstrated that in CD38-/- CAMs, 7-Ket failed to stimulate the production of O2-., while in CD38+/+ CAMs 7-Ket induced marked O2-. production and enhancement of Nrf2 activity, which was substantially attenuated by NOX4 gene silencing. Finally, we demonstrated that 7-Ket-induced and NOX4-dependent O2-. production was inhibited by 8-Br-cADPR, an antagonist of cADPR or NED-19, an antagonist of NAADP as product of CD38 ADP-ribosylcyclase, which significantly inhibited the level of cytosolic Ca2+ and the activation of Nrf2 under 7-Ket. Conclusion

  15. Lack of effect of cell-wall targeted antibacterials on biofilm formation and antifungal susceptibility of Candidaspecies

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    Gisela Myrian de Lima Leite

    2014-09-01

    Full Text Available The use of central venous catheters (CVC and broad-spectrum antibacterials are among the main risk factors for the development of candidemia in patients admitted to intensive care units (ICU. It is known that some antibacterials increase the resistance of these yeasts to azole antifungals. Thus, the aim of this research was to determine whether yeast present in CVC colonizations previously exposed to cell-wall targeted antibacterials benefit from a reduction in susceptibility to fluconazole and voriconazole, facilitating their ability to form biofilms. Candida albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. guilhermondii were seeded into antibacterial (cefepime, meropenem, vancomycin, and piperacillin-tazobactam gradient plates produced in Mueller-Hinton Agar. The susceptibility to fluconazole and voriconazole and the biofilm formation of the yeasts were tested before and after exposure to the antibacterials. None of the antibacterials exerted a significant effect on the in vitro susceptibility of the yeasts to the antifungal agents or on their ability to form biofilms. These results suggest that increased candidemia in ICU patients is not attributable to possible alterations in the yeasts, but is more likely caused by a weakening of the patient's general condition after long exposure to infection.

  16. Nonobese Diabetic (NOD Mice Lack a Protective B-Cell Response against the “Nonlethal” Plasmodium yoelii 17XNL Malaria Protozoan

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    Mirian Mendoza

    2016-01-01

    Full Text Available Background. Plasmodium yoelii 17XNL is a nonlethal malaria strain in mice of different genetic backgrounds including the C57BL/6 mice (I-Ab/I-Enull used in this study as a control strain. We have compared the trends of blood stage infection with the nonlethal murine strain of P. yoelii 17XNL malaria protozoan in immunocompetent Nonobese Diabetic (NOD mice prone to type 1 diabetes (T1D and C57BL/6 mice (control mice that are not prone to T1D and self-cure the P. yoelii 17XNL infection. Prediabetic NOD mice could not mount a protective antibody response to the P. yoelii 17XNL-infected red blood cells (iRBCs, and they all succumbed shortly after infection. Our data suggest that the lack of anti-P. yoelii 17XNL-iRBCs protective antibodies in NOD mice is a result of parasite-induced, Foxp3+ T regulatory (Treg cells able to suppress the parasite-specific antibody secretion. Conclusions. The NOD mouse model may help in identifying new mechanisms of B-cell evasion by malaria parasites. It may also serve as a more accurate tool for testing antimalaria therapeutics due to the lack of interference with a preexistent self-curing mechanism present in other mouse strains.

  17. Immunohistochemical markers of advanced basal cell carcinoma: CD56 is associated with a lack of response to vismodegib.

    Science.gov (United States)

    Castillo, Jean-Marie; Knol, Anne-Chantal; Nguyen, Jean-Michel; Khammari, Amir; Saint-Jean, Mélanie; Dreno, Brigitte

    2016-10-01

    Vismodegib is an effective treatment for advanced basal cell carcinoma (BCC), but primary resistance to vismodegib remains to be elucidated. Alternative approaches are warranted to help selecting patients most likely to be responsive to treatment. The identification of immunohistochemical markers may support this perspective, as well as better understanding of resistance mechanisms. To determine the level of expression of CD56, PDGF-R, CD117, MMP9, TIMP3, and CXCR4 in advanced BCC, and explore whether expression levels are associated with non-response to vismodegib. A cross-sectional study was conducted. Immunohistochemical markers were selected based on their roles in tumour proliferation and/or migration in skin tumours. Tissue samples included pretreatment advanced BCC samples from patients treated with vismodegib, with an available response after six months of treatment. Regression optimised models were used to build hypotheses regarding a possible association between expression levels and non-response to vismodegib, which was then tested by logistic regression. Twenty-three patients were included. The percentage of samples expressing markers ranged from 43.5% (CD117) to 91.3% (CXCR4). CD56 expression was significantly associated with an increased risk of non-response to vismodegib (OR = 5.5; CI 95%: 3.4-29.8; p = 0.0488); a similar association was suggested for CXCR4 (p = 0.066), but not identified for other markers. These results provide a better understanding of the expression of immunohistochemical markers in advanced BCC. Further detailed analysis of CD56 expression may provide insights into guiding further investigation of the correlation between this marker and non-response to vismodegib.

  18. Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations.

    Science.gov (United States)

    Wang, Yun; Woodgate, Roger; McManus, Terrence P; Mead, Samantha; McCormick, J Justin; Maher, Veronica M

    2007-04-01

    Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to UV-induced mutations, and ultimately their malignant transformation.

  19. Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

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    Strain Alastair J

    2010-03-01

    Full Text Available Abstract The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%, and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC derived from human umbilical cord blood (UCB and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB.

  20. Inability to induce consistent T-cell responses recognizing conserved regions within HIIV-1 antigens: a potential mechanism for lack of vaccine efficacy in the step study

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    Korber, Bette [Los Alamos National Laboratory; Szinger, James [Los Alamos National Laboratory

    2009-01-01

    T cell based vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a high probability of matching the epitope induced by vaccination with the infecting viral strain. We compared the frequency and specificity of the CTL epitopes elicited by the replication defective AdS gag/pol/nef vaccine used in the STEP trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. On average vaccination elicited only one epitope per gene. Importantly, the highly conserved epitopes in gag, pol, and nef (> 80% of strains in the current collection of the Los Alamos database [www.hiv.lanl.gov]) were rarely elicited by vaccination. Moreover there was a statistically significant skewing of the T cell response to relative variable epitopes of each gene; only 20% of persons possessed > 3 T cell responses to epitopes likely to be found in circulating strains in the CladeB populations in which the Step trial was conducted. This inability to elicit T cell responses likely to be found in circulating viral strains is a likely factor in the lack of efficacy of the vaccine utilized in the STEP trial. Modeling of the epitope specific responses elicited by vaccination, we project that a median of 8-10 CD8+ T cell epitopes are required to provide >80% likelihood of eliciting at least 3 CD8+ T cell epitopes that would be found on a circulating population of viruses. Development of vaccine regimens which elicit either a greater breadth of responses or elicit responses to conserved regions of the HIV-1 genome are needed to fully evaluate the concept of whether induction of T cell immunity can alter HIV-1 in vivo.

  1. Benzene activates caspase-4 and -12 at the transcription level, without an association with apoptosis, in mouse bone marrow cells lacking the p53 gene

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Jung-Yeon; Han, Jeong-Hee; Yoon, Byung-Il [Kangwon National University, School of Veterinary Medicine, Chuncheon, Gangwon (Korea); Hirabayashi, Yoko; Kodama, Yukio; Kanno, Jun [National Institute of Health Sciences, Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, Tokyo (Japan); Choi, Yang-Kyu [Konkuk University, College of Veterinary Medicine, Seoul (Korea); Inoue, Tohru [National Institute of Health Sciences, Biological Safety and Research Center, Tokyo (Japan)

    2009-08-15

    Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. However, although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying lympho/leukemogenicity is not fully understood. Caspase-4 (alias caspase-11) and -12 are inflammatory caspases implicated in inflammation and endoplasmic reticulum stress-induced apoptosis. The objectives of this study were to investigate the altered expression of caspase-4 and -12 in mouse bone marrow after benzene exposure and to determine whether their alterations are associated with benzene-induced bone marrow toxicity, especially cellular apoptosis. In addition, we evaluated whether the p53 gene is involved in regulating the mechanism, using both wild-type (WT) mice and mice lacking the p53 gene. For this study, 8-week-old C57BL/6 mice [WT and p53 knockout (KO)] were administered a benzene solution (150 mg/kg diluted in corn oil) via oral gavage once daily, 5 days/week, for 1 or 2 weeks. Blood and bone marrow cells were collected and cell counts were measured using a Coulter counter. Total mRNA and protein extracts were prepared from the harvested bone marrow cells. Then qRT-PCR and Western blotting were performed to detect changes in the caspases at the mRNA and protein level, respectively. A DNA fragmentation assay and Annexin-V staining were carried out on the bone marrow cells to detect apoptosis. Results indicated that when compared to the control, leukocyte number and bone marrow cellularity decreased significantly in WT mice. The expression of caspase-4 and -12 mRNA increased significantly after 12 days of benzene treatment in the bone marrow cells of benzene-exposed p53KO mice. However, apoptosis detection assays indicated no evidence of apoptosis in p53KO or WT mice. In addition, no changes of other apoptosis-related caspases, such as caspase-3 and -9, were found in WT or p53KO mice at the

  2. A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

    Science.gov (United States)

    Jones, Tim D.; Hearn, Arron R.; Holgate, Robert G.E.; Kozub, Dorota; Fogg, Mark H.; Carr, Francis J.; Baker, Matthew P.; Lacadena, Javier; Gehlsen, Kurt R.

    2016-01-01

    Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics. PMID:27578884

  3. Doxycycline-Regulated p16(MTS1) Expression Suppresses the Anchorage-Independence and Tumorigenicity of Breast Cancer Cell Lines that Lack Endogenous p16.

    Science.gov (United States)

    Todd, Maria C; Langan, Thomas A; Sclafani, Robert A

    2017-01-01

    The RB pathway controls the critical transition from G1 into S phase of the mammalian cell cycle. Deregulation of the RB pathway by means of RB or p16 inactivation has been implicated in the development of virtually all human cancers. Such findings have led to the view that the loss of RB-mediated regulation at the G1/S checkpoint is a precondition for human malignancy. Our analysis of the RB-positive MCF-7 and ZR75.1 breast cancer cell lines revealed a lack of endogenous p16 protein expression as a result of the homozygous deletion and methylation of the p16 gene at the CDKN2A locus, respectively. We employed the TET-OFF inducible expression system to investigate the effects of non-growth inhibitory levels of functional p16 protein upon the in vitro and in vivo transformed properties of the MCF-7 and ZR75.1 cell lines. Stable transfectants of MCF-7 and ZR75.1 cells were isolated that expressed different levels of p16 protein in the absence of doxycycline (DOX) but continued to proliferate in culture. Transfectants that expressed modest levels of p16 (relative to SV40 T antigen-transformed HBL-100 breast epithelial cells) demonstrated a marked suppression of anchorage-independent growth in soft agar. Further, the induction of moderate and high levels of p16 (relative to HBL-100) resulted in the suppression of tumorigenicity of both MCF-7 and ZR75.1 cells as assayed by injection into nude mice. From these data, we concluded that RB pathway restoration by non-growth inhibitory levels of p16 protein was sufficient to revert breast cancer cells to a non-transformed and non-tumorigenic state.

  4. Doxycycline-Regulated p16MTS1 Expression Suppresses the Anchorage-Independence and Tumorigenicity of Breast Cancer Cell Lines that Lack Endogenous p16

    Science.gov (United States)

    Todd, Maria C; Langan, Thomas A; Sclafani, Robert A

    2017-01-01

    The RB pathway controls the critical transition from G1 into S phase of the mammalian cell cycle. Deregulation of the RB pathway by means of RB or p16 inactivation has been implicated in the development of virtually all human cancers. Such findings have led to the view that the loss of RB-mediated regulation at the G1/S checkpoint is a precondition for human malignancy. Our analysis of the RB-positive MCF-7 and ZR75.1 breast cancer cell lines revealed a lack of endogenous p16 protein expression as a result of the homozygous deletion and methylation of the p16 gene at the CDKN2A locus, respectively. We employed the TET-OFF inducible expression system to investigate the effects of non-growth inhibitory levels of functional p16 protein upon the in vitro and in vivo transformed properties of the MCF-7 and ZR75.1 cell lines. Stable transfectants of MCF-7 and ZR75.1 cells were isolated that expressed different levels of p16 protein in the absence of doxycycline (DOX) but continued to proliferate in culture. Transfectants that expressed modest levels of p16 (relative to SV40 T antigen-transformed HBL-100 breast epithelial cells) demonstrated a marked suppression of anchorage-independent growth in soft agar. Further, the induction of moderate and high levels of p16 (relative to HBL-100) resulted in the suppression of tumorigenicity of both MCF-7 and ZR75.1 cells as assayed by injection into nude mice. From these data, we concluded that RB pathway restoration by non-growth inhibitory levels of p16 protein was sufficient to revert breast cancer cells to a non-transformed and non-tumorigenic state.

  5. Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies.

    Science.gov (United States)

    Engelhardt, Othmar G; Sirma, Hüseyin; Pandolfi, Pier-Paolo; Haller, Otto

    2004-08-01

    The interferon-induced murine Mx1 GTPase is a nuclear protein. It specifically inhibits influenza A viruses at the step of primary transcription, a process known to occur in the nucleus of infected cells. However, the exact mechanism of inhibition is still poorly understood. The Mx1 GTPase has previously been shown to accumulate in distinct nuclear dots that are spatially associated with promyelocytic leukaemia protein (PML) nuclear bodies (NBs), but the significance of this association is not known. Here it is reported that, in cells lacking PML and, as a consequence, PML NBs, Mx1 still formed nuclear dots. These dots were indistinguishable from the dots observed in wild-type cells, indicating that intact PML NBs are not required for Mx1 dot formation. Furthermore, Mx1 retained its antiviral activity against influenza A virus in these PML-deficient cells, which were fully permissive for influenza A virus. Nuclear Mx proteins from other species showed a similar subnuclear distribution. This was also the case for the human MxA GTPase when this otherwise cytoplasmic protein was translocated into the nucleus by virtue of a foreign nuclear localization signal. Human MxA and mouse Mx1 do not interact or form heterooligomers. Yet, they co-localized to a large degree when co-expressed in the nucleus. Taken together, these findings suggest that Mx1 dots represent distinct nuclear domains ('Mx nuclear domains') that are frequently associated with, but functionally independent of, PML NBs.

  6. Synergistic inhibition of the APC/C by the removal of APC15 in HCT116 cells lacking UBE2C

    Science.gov (United States)

    Garvanska, Dimitriya H.; Larsen, Marie Sofie Yoo

    2016-01-01

    ABSTRACT The spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly, in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components. PMID:27591192

  7. T-cell-rich large B-cell lymphoma. A study of 30 cases, supporting its histologic heterogeneity and lack of clinical distinctiveness.

    Science.gov (United States)

    Krishnan, J; Wallberg, K; Frizzera, G

    1994-05-01

    To determine whether correlations existed between morphologic and immunophenotypic findings and clinical characteristics, 30 cases of T-cell-rich large B-cell lymphomas (TBL) were evaluated by histopathology, immunostaining, and polymerase chain reaction on paraffin-embedded material. All were characterized by a polymorphic cell composition, including a variable mixture of small and large lymphoid cells and reactive cell. Most cases (87%) fitted into one of three main histologic types of non-Hodgkin's lymphoma (diffuse, mixed cell; diffuse, large cell; follicular and diffuse, mixed cell), and one group of eight cases had the prototypic features described by Ramsay et al. (17). All cases showed a component of large CD20(L26)+ MB2+ B cells in a predominant back-ground of reactive T cells (> 50% of the total lymphoid forms). Clonality was demonstrated by light chain restriction in 67% of cases and by rearrangement of the immunoglobulin heavy chain gene and bcl-2 gene in 64% and 28% of cases, respectively. The patients were predominantly men (70%), ages 18-83 years (median of 62.5), and were initially seen predominantly with nodal disease (and extranodal involvement in 20%) at advanced stages (III-IV: 77%). Treatment was mostly aggressive chemotherapy, and the outcomes were favorable (84% alive and well). These features are not distinctive as compared with those of typical large-cell lymphoma, nor did subgroups within the series (prototypic cases versus others; cases with less [ 70%] T-cell infiltration) significantly differ in clinical presentation or outcome. Thus, this study confirms that TBL, while useful as a diagnostic variant to be distinguished from both peripheral T-cell lymphoma and Hodgkin's disease, is a heterogeneous assortment of diverse histopathologic categories rather than a clinicopathologic entity. The term "T-cell rich" might, however, be usefully retained as a morphologic specification to be added to recognized histologic categories of lymphoma.

  8. Lack of estrogen down-regulates CXCR4 expression on Treg cells and reduces Treg cell population in bone marrow in OVX mice.

    Science.gov (United States)

    Fan, X-L; Duan, X-B; Chen, Z-H; Li, M; Xu, J-S; Ding, G-M

    2015-05-08

    Postmenopausal osteoporosis (PMO) is the most common metabolic bone disease in women after menopausal. Recent works focused on cross—talk between immune regulation and bone metabolism pathways and suggested Treg cells suppressed bone resorption and osteoclasts (OC) differentiation in bone marrow via cell—cell contact interaction and/or secreting of IL—10 and TGF—beta. In this study, we investigated the impact of estrogen on regulatory T cells (Treg cells) trafficking and staying in bone marrow and we found that a significant reduction of Treg cell population in bone marrow in estrogen deficiency ovariectomied (OVX) mice. We then studied the expressions of chemokines CXCL12/CXCR4 axes, which were critical to Treg cells migration and our data show the expression of CXCR4 on Treg cells was relative with oestrogen in vivo, however, the expression of CXCL12 was not. Furthermore, the loss of trafficking ability of Treg cells in OVX mice was recoverable in our system. These findings may mechanistically explain why Treg cells lose their suppressive functions on the regulation of OC cells and demonstrate a previously unappreciated role for estrogen, which may be critical to the novel therapy in clinical practice of PMO patients.

  9. Lack of Suppressive CD4+CD25+FOXP3+ T Cells in Advanced Stages of Primary Cutaneous T-Cell Lymphoma

    OpenAIRE

    Tiemessen, Machteld M.; Mitchell, Tracey J.; Hendry, Lisa; Whittaker, Sean J; Taams, Leonie S.; John, Susan

    2006-01-01

    Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapabl...

  10. Verrucous carcinomas of the head and neck, including those with associated squamous cell carcinoma, lack transcriptionally active high-risk human papillomavirus.

    Science.gov (United States)

    Patel, Kalyani R; Chernock, Rebecca D; Zhang, Tian R; Wang, Xiaowei; El-Mofty, Samir K; Lewis, James S

    2013-11-01

    Most oropharyngeal squamous cell carcinomas (SCC) and histologic variants harbor transcriptionally active human papillomavirus (HPV). While HPV DNA can be found in many non-oropharyngeal head and neck carcinomas, transcriptionally active HPV is rare. Verrucous carcinoma is a variant with bland cytology, warty appearance, locally destructive growth, and lack of metastasis when lacking a frankly invasive carcinoma component. Studies have shown variable rates of HPV DNA and p16 protein expression in such tumors but still have not clearly addressed if the virus has biological activity or clinical relevance in the positive cases. Department files were searched for verrucous neoplasms, including pure verrucous carcinoma, verrucous carcinoma with dysplasia or minimal invasion, and SCC arising in verrucous carcinoma (ie, having a major component of frankly invasive carcinoma). p16 immunohistochemistry, HPV DNA polymerase chain reaction (PCR) and E6/E7 mRNA reverse transcription PCR for high-risk HPV types were performed. Of the 49 cases, 6 (12.2%) showed strong (>50%) staining for p16. HPV DNA was detected in 7/49 (14.3%) cases, but only one case was positive for both p16, and HPV DNA. A total of 36 cases yielded sufficient RNA for RT-PCR (18 verrucous carcinomas, 13 atypical verrucous carcinomas, and 5 SCC arising in verrucous carcinoma). All 36 were negative, including the four p16-positive and three HPV DNA-positive tumors tested. Although a minority of verrucous carcinoma lesions are p16 and HPV DNA positive, transcriptionally active high-risk HPV is uniformly absent. These findings argue that verrucous carcinoma and its related squamous cell carcinomas are not HPV-driven tumors.

  11. Lack of nonfunctional B-cell receptor rearrangements in a patient with normal B cell numbers despite partial RAG1 deficiency and atypical SCID/Omenn syndrome

    DEFF Research Database (Denmark)

    Ohm-Laursen, Line; Nielsen, Christian; Fisker, Niels

    2008-01-01

    and the patient had eosinophilia. These presentations are consistent with atypical severe combined immunodeficiency (SCID)/Omenn Syndrome and the diagnosis was confirmed by demonstration of homozygosity for the R841W mutation in the catalytic core of RAG1. Comparison of the patient's immunoglobulin heavy chain...... chromosome 14. CONCLUSION: We hypothesize that the R841W mutation causes a malfunction of RAG1 that has differential outcome on V(D)J recombination in B and T cells, as the patient had normal B cell numbers but suffered severe alpha-beta T-cell immunodeficiency.......INTRODUCTION: A 2.5-month old boy presented with recurrent wheezing, protracted diarrhea, erythrodermia, and failure to thrive. METHODS AND RESULTS: Laboratory analysis showed lymphocytopenia with severely reduced T-cell numbers but normal numbers of B and NK cells. Serum IgE was increased...

  12. Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems.

    Science.gov (United States)

    Kim, Woe Yeon; Lee, Sun Yong; Jung, Young Jun; Chae, Ho Byoung; Nawkar, Ganesh M; Shin, Mi Rim; Kim, Sun Young; Park, Jin Ho; Kang, Chang Ho; Chi, Yong Hun; Ahn, Il Pyung; Yun, Dae Jin; Lee, Kyun Oh; Kim, Young-Myeong; Kim, Min Gab; Lee, Sang Yeol

    2011-12-09

    A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

  13. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    Science.gov (United States)

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  14. Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

    Directory of Open Access Journals (Sweden)

    Nyambayar Dashtsoodol

    Full Text Available Invariant Vα14 natural killer T (NKT cells, characterized by the expression of a single invariant T cell receptor (TCR α chain encoded by rearranged Trav11 (Vα14-Traj18 (Jα18 gene segments in mice, and TRAV10 (Vα24-TRAJ18 (Jα18 in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

  15. Cytokine production but lack of proliferation in peripheral blood mononuclear cells from chronic Chagas' disease cardiomyopathy patients in response to T. cruzi ribosomal P proteins.

    Directory of Open Access Journals (Sweden)

    Silvia A Longhi

    2014-06-01

    Full Text Available BACKGROUND: Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC. It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC stimulated with P2β, the C-terminal portion of P0 (CP0 proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells. CONCLUSIONS/SIGNIFICANCE: Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T

  16. Alloactivated HLA class II-positive T-cell lines induce IL-2 reactivity but lack accessory cell function in mixed leukocyte culture

    DEFF Research Database (Denmark)

    Odum, N; Dickmeiss, E; Hofmann, B

    1989-01-01

    ). From 70 to 90% of the Ta were HLA class II-positive as judged by the reactions with HLA class II-reactive monoclonal antibodies, and the Ta carried the DR allospecificities of the original T-cell donor when typed in the microcytotoxic test using DR-specific alloantisera. Neither irradiated nor...

  17. Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease.

    Directory of Open Access Journals (Sweden)

    Wenjian Ma

    2011-04-01

    Full Text Available DNA double-strand breaks (DSBs are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.

  18. Lack of galectin-3 increases Jagged1/Notch activation in bone marrow-derived dendritic cells and promotes dysregulation of T helper cell polarization.

    Science.gov (United States)

    Fermino, Marise L; Dylon, L Sebastian D; Cecílio, Nerry T; Santos, Sofia N; Toscano, Marta A; Dias-Baruffi, Marcelo; Roque-Barreira, Maria C; Rabinovich, Gabriel A; Bernardes, Emerson S

    2016-08-01

    Galectin-3, an endogenous glycan-binding protein, is abundantly expressed at sites of inflammation and immune cell activation. Although this lectin has been implicated in the control of T helper (Th) polarization, the mechanisms underlying this effect are not well understood. Here, we investigated the role of endogenous galectin-3 during the course of experimental Leishmania major infection using galectin-3-deficient (Lgals3(-/-)) mice in a BALB/c background and the involvement of Notch signaling pathway in this process. Lgals3(-/-) mice displayed an augmented, although mixed Th1/Th2 responses compared with wild-type (WT) mice. Concomitantly, lymph node and footpad lesion cells from infected Lgals3(-/-) mice showed enhanced levels of Notch signaling components (Notch-1, Jagged1, Jagged2 and Notch target gene Hes-1). Bone marrow-derived dendritic cells (BMDCs) from uninfected Lgals3(-/-) mice also displayed increased expression of the Notch ligands Delta-like-4 and Jagged1 and pro-inflammatory cytokines. In addition, activation of Notch signaling in BMDCs upon stimulation with Jagged1 was more pronounced in Lgals3(-/-) BMDCs compared to WT BMDCs; this condition resulted in increased production of IL-6 by Lgals3(-/-) BMDCs. Finally, addition of exogenous galectin-3 to Lgals3(-/-) BMDCs partially reverted the increased sensitivity to Jagged1 stimulation. Our results suggest that endogenous galectin-3 regulates Notch signaling activation in BMDCs and influences polarization of T helper responses, thus increasing susceptibility to L. major infection.

  19. Lack of significant skin inflammation during elimination by apoptosis of large numbers of mouse cutaneous mast cells after cessation of treatment with stem cell factor.

    Science.gov (United States)

    Maurer, Marcus; Galli, Stephen J

    2004-12-01

    We previously reported that subcutaneous (s.c.) administration of stem cell factor (SCF), the ligand for the c-Kit receptor, to the back skin of mice promotes marked local increases in the numbers of cutaneous mast cells (MCs), and that cessation of SCF treatment results in the rapid reduction of cutaneous MC populations by apoptosis. In the present study, we used the 125I-fibrin deposition assay, a very sensitive method for quantifying increased vascular permeability, to assess whether the clearance of large numbers of apoptotic MCs is associated with significant cutaneous inflammation. The s.c. injection of rrSCF164 (30 or 100 microg/kg/day) or rrSCF164-peg (polyethylene glycol-treated SCF, 30 or 100 microg/kg/day) for 23 days increased the numbers of dermal MCs at skin injection sites from 5.1+/-0.7 MCs/mm2 to 36.4+/-4.1, 34.7+/-9.7, 52.5+/-5.8, and 545+/-97 MCs/mm2, respectively. In contrast, MC numbers were markedly lower in mice that had been treated with SCF for 21 days, followed by 2 days of injection with the vehicle alone. Notably, when tested during the period of rapid reduction of skin MCs,125I-fibrin deposition in the skin was very similar to that in mice receiving continuous treatment with SCF or vehicle. We conclude that the rapid elimination of even very large populations of MCs by apoptosis, which also results in the clearance of the considerable quantities of proinflammatory products stored by these cells, does not lead to significant local cutaneous inflammatory responses.

  20. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  1. Staphylococcus aureus mutants lacking cell wall-bound protein A found in isolates from bacteraemia, MRSA infection and a healthy nasal carrier.

    Science.gov (United States)

    Sørum, Marit; Sangvik, Maria; Stegger, Marc; Olsen, Renate S; Johannessen, Mona; Skov, Robert; Sollid, Johanna U E

    2013-02-01

    Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.

  2. Intrahepatic B-cell follicles of chronically hepatitis C virus-infected individuals lack signs of an ectopic germinal center reaction.

    Science.gov (United States)

    Tucci, Felicia A; Broering, Ruth; Lutterbeck, Melanie; Schlaak, Joerg F; Küppers, Ralf

    2014-06-01

    Chronic infection with hepatitis C virus (HCV) often affects the B-cell compartment, leading to the occurrence of autoimmunity and B-cell lymphoproliferation, in particular mixed cryoglobulinemia and B-cell lymphomas. HCV presumably causes these lymphoproliferations by chronic antigenic stimulation and/or direct mutagenic effects on B cells. It has been speculated that the interaction of HCV with B cells and the expansion of antigen-triggered B cells happens in germinal center-like structures in the livers of HCV carriers. We studied rearranged immunoglobulin V(H) genes from seven B-cell follicles microdissected from the livers of three unselected chronic HCV patients. The follicles consisted of polyclonal naive and memory B-cell populations with only rare indication of minor clonal expansions and no evidence for active somatic hypermutation. Frequent detection of V(H) rearrangements using the VH1-69 gene segment nevertheless indicated that at least a fraction of the B cells is HCV-specific and/or autoreactive. Thus, the typical intrahepatic B-cell follicles in chronic HCV carriers do not function as ectopic germinal centers for clonal expansion and affinity maturation of B cells. Hence, autoreactive and HCV-specific B-cell clones might either develop in secondary lymphoid organs or in intrahepatic follicles only under particular, yet undefined, circumstances.

  3. Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

    Science.gov (United States)

    Naeser, F K; Barfod, N M; Bichel, P

    1978-02-14

    In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.

  4. Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

    Science.gov (United States)

    Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFNgamma). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a dis...

  5. Lack of Rb and p53 delays cerebellar development and predisposes to large cell anaplastic medulloblastoma through amplification of N-Myc and Ptch2.

    Science.gov (United States)

    Shakhova, Olga; Leung, Carly; van Montfort, Erwin; Berns, Anton; Marino, Silvia

    2006-05-15

    Medulloblastomas are among the most common malignant brain tumors in childhood. They typically arise from neoplastic transformation of granule cell precursors in the cerebellum via deregulation of molecular pathways involved in normal cerebellar development. In a mouse model, we show here that impairment of the balance between proliferation and differentiation of granule cell precursors in the external granular layer of the developing cerebellum predisposes but is not sufficient to induce neoplastic transformation of these progenitor cells. Using array-based chromosomal comparative genomic hybridization, we show that genetic instability resulting from inactivation of the p53 pathway together with deregulation of proliferation induced by Rb loss eventually leads to neoplastic transformation of these cells by acquiring additional genetic mutations, mainly affecting N-Myc and Ptch2 genes. Moreover, we show that p53 loss influences molecular mechanisms that cannot be mimicked by the loss of either p19(ARF), p21, or ATM.

  6. Synergistic inhibition of the APC/C by the removal of APC15 in HCT116 cells lacking UBE2C

    DEFF Research Database (Denmark)

    Garvanska, Dimitriya H; Larsen, Marie Sofie Yoo; Nilsson, Jakob

    2016-01-01

    that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor...

  7. Lack of sik1 in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2.

    Directory of Open Access Journals (Sweden)

    Antonio Romito

    Full Text Available Sik1 (salt inducible kinase 1 is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57(Kip2 is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation.

  8. Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells

    Energy Technology Data Exchange (ETDEWEB)

    Reinhard, J.F. Jr.; Diliberto, E.J. Jr.; Viveros, O.H.; Daniels, A.J.

    1987-11-01

    Cultures of bovine adrenomedullary chromaffin cells accumulated 1-methyl-4-phenylpyridinium (MPP/sup +/) in a time- and concentration-dependent manner by a process that was prevented by desmethylimipramine. The subcellular localization of the incorporated (methyl-/sup 3/H)MPP/sup +/ was examined by differential centrifugation and sucrose density gradient fractionation and was found to be predominantly colocalized with catecholamines in chromaffin vesicles, and negligible amounts were detected within the mitochondrial fraction. When chromaffin cell membranes were made permeable with the detergent digitonin the absence of calcium, there was no increase in the release of (/sup 3/H)MPP/sup +/, indicating that there is negligible accumulation of the neurotoxin in the cytosol. Simultaneous exposure to digitonin and calcium induced cosecretion of MPP/sup +/ and catecholamines. Stimulation of the cells with nicotine released both catecholamines and MPP/sup +/ at identical rates and percentages of cellular content in a calcium-dependent manner. Last, when cells were incubated with MPP/sup +/ in the presence of tetrabenazine (an inhibitor of vesicular uptake), the chromaffin cell toxicity of MPP/sup +/ was potentiated. The authors submit that the ability of the chromaffin cells to take up and store MPP/sup +/ in the chromaffin vesicle prevents the toxin's interaction with other structures and, thus, prevents cell damage. As an extension of this hypothesis, the relative resistance of some brain monoaminergic neurons to the toxic actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may result from the subcellular sequestration of MPP/sup +/ in the storage vesicle.

  9. Gene expression profiles in mouse embryo fibroblasts lacking stathmin, a microtubule regulatory protein, reveal changes in the expression of genes contributing to cell motility

    Directory of Open Access Journals (Sweden)

    Cassimeris Lynne

    2009-07-01

    Full Text Available Abstract Background Stathmin (STMN1 protein functions to regulate assembly of the microtubule cytoskeleton by destabilizing microtubule polymers. Stathmin over-expression has been correlated with cancer stage progression, while stathmin depletion leads to death of some cancer cell lines in culture. In contrast, stathmin-null mice are viable with minor axonopathies and loss of innate fear response. Several stathmin binding partners, in addition to tubulin, have been shown to affect cell motility in culture. To expand our understanding of stathmin function in normal cells, we compared gene expression profiles, measured by microarray and qRT-PCR, of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1-/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin. Results Microarray analysis of STMN1 loss at a fold change threshold of ≥ 2.0 revealed expression changes for 437 genes, of which 269 were up-regulated and 168 were down-regulated. Microarray data and qRT-PCR analysis of mRNA expression demonstrated changes in the message levels for STMN4, encoding RB3, a protein related to stathmin, and in alterations to many tubulin isotype mRNAs. KEGG Pathway analysis of the microarray data indicated changes to cell motility-related genes, and qRT-PCR plates specific for focal adhesion and ECM proteins generally confirmed the microarray data. Several microtubule assembly regulators and motors were also differentially regulated in STMN1-/- cells, but these changes should not compensate for loss of stathmin. Conclusion Approximately 50% of genes up or down regulated (at a fold change of ≥ 2 in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct

  10. Brain lesions comprised of aluminum-rich cells that lack microtubules may be associated with the cognitive deficit of Alzheimer's disease.

    Science.gov (United States)

    Walton, J R

    2009-11-01

    A recent longitudinal study described an inducible rodent model for age-related cognitive deterioration. This model was produced by chronically feeding rats aluminum, from age 12 months onwards, in measured amounts equivalent to total aluminum levels ingested by Americans from their food, beverages and aluminum additives. The rats performed a hippocampal-dependent spatial memory discrimination task weekly throughout middle age and old age. One-third of the rats attained significantly lower mean performance scores in old age than middle age, in an aluminum dose-dependent manner, and exhibited behavioral signs observed in dementia. The present study used histological and immunohistochemical techniques to identify neuropathological difference between brains of rats that showed cognitive deterioration and the cognitively intact controls. Most aged rat brains had large numbers of aluminum-loaded pyramidal cells in their entorhinal cortex and temporal association cortex but the cognitively deteriorated rats had threefold more such cells than controls (paluminum-rich microtubule-depleted pyramidal cells with shriveled processes, and loss of synapse density. Corticolimbic sections from brains of humans with Alzheimer's disease also showed neuropathology consistent with this type of damage. The evidence suggests bioavailable aluminum gradually accumulates in cortical and limbic regions of susceptible subjects' brains, eventually producing hippocampal lesions consisting of dysfunctional aluminum-rich microtubule-depleted pyramidal cells with damaged neurites and synapse loss. These lesions expand over time, disrupting afferent and efferent hippocampal circuitry with the development of clinically overt dementia.

  11. Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

    Science.gov (United States)

    Ganapathy, Suthakar; Fagman, Johan B; Shen, Ling; Yu, Tianqi; Zhou, Xiaodong; Dai, Wei; Makriyannis, Alexandros; Chen, Changyan

    2016-12-20

    Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

  12. Lack of TIMP-1 tumour cell immunoreactivity predicts effect of adjuvant anthracycline-based chemotherapy in patients (n=647) with primary breast cancer

    DEFF Research Database (Denmark)

    Willemoe, Gro L.; Hertel, Pernille Bræmer; Bartels, Annette;

    2009-01-01

    PURPOSE: A number of prospective studies have shown that adjuvant CEF significantly improves disease-free and overall survival as compared to CMF in breast cancer patients. Our aim was to determine whether the benefit of epirubicin versus methotrexate differs according to TIMP-1 tumour cell...

  13. Lung metastasis fails in MMTV-PyMT oncomice lacking S100A4 due to a T-cell deficiency in primary tumors

    DEFF Research Database (Denmark)

    Grum-Schwensen, Birgitte; Klingelhöfer, Jörg; Grigorian, Mariam

    2010-01-01

    Interactions between tumor and stroma cells are essential for the progression of cancer from its initial growth at a primary site to its metastasis to distant organs. The metastasis-stimulating protein S100A4 exerts its function as a stroma cell-derived factor. Genetic depletion of S100A4...... significantly reduced the metastatic burden in lungs of PyMT-induced mammary tumors. In S100A4(+/+) PyMT mice, massive leukocyte infiltration at the site of the growing tumor at the stage of malignant transition was associated with increased concentration of extracellular S100A4 in the tumor microenvironment....... In contrast, in S100A4(-/-) PyMT tumors, a significant suppression of T-cell infiltration was documented at the transition period. In vitro, the S100A4 protein mediated the attraction of T cells. Moreover, S100A4(+/+), but not S100A4(-/-), fibroblasts stimulated the invasion of T lymphocytes into fibroblast...

  14. A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses.

    Science.gov (United States)

    García-Arriaza, Juan; Nájera, José Luis; Gómez, Carmen E; Tewabe, Nolawit; Sorzano, Carlos Oscar S; Calandra, Thierry; Roger, Thierry; Esteban, Mariano

    2011-01-01

    The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.

  15. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra

    Energy Technology Data Exchange (ETDEWEB)

    Vreeswijk, Maaike P.G., E-mail: vreeswijk@lumc.nl [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands); Department of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Building 2, Postzone S-04, P.O. Box 9600, 2300 RC Leiden (Netherlands); Meijers, Caro M.; Giphart-Gassler, Micheline; Vrieling, Harry; Zeeland, Albert A. van; Mullenders, Leon H.F.; Loenen, Wil A.M. [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands)

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  16. Lack of effects of typical and atypical antipsychotics in DARPP-32 and NCS-1 levels in PC12 cells overexpressing NCS-1

    Directory of Open Access Journals (Sweden)

    Reis Helton J

    2010-06-01

    Full Text Available Abstract Background Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC: Neuronal Calcium Sensor-1 (NCS-1 and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT and PC12 cells stably overexpressing NCS-1 (PC12 Clone with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment. Results We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol or atypical (Clozapine and Risperidone antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments. Conclusions Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.

  17. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra.

    Science.gov (United States)

    Vreeswijk, Maaike P G; Meijers, Caro M; Giphart-Gassler, Micheline; Vrieling, Harry; van Zeeland, Albert A; Mullenders, Leon H F; Loenen, Wil A M

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4 PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C>T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  18. Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

    Directory of Open Access Journals (Sweden)

    Dale Christiansen

    2008-07-01

    Full Text Available The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3, is believed to be critical for natural killer T (NKT cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S. We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3Gal xenoantigen, with obvious significance in the field of xenotransplantation.

  19. Embryonic lethality in mice lacking the nuclear factor of activated T cells 5 protein due to impaired cardiac development and function.

    Directory of Open Access Journals (Sweden)

    Man Chi Mak

    Full Text Available Nuclear factor of activated T cells 5 protein (NFAT5 is thought to be important for cellular adaptation to osmotic stress by regulating the transcription of genes responsible for the synthesis or transport of organic osmolytes. It is also thought to play a role in immune function, myogenesis and cancer invasion. To better understand the function of NFAT5, we developed NFAT5 gene knockout mice. Homozygous NFAT5 null (NFAT5(-/- mouse embryos failed to develop normally and died after 14.5 days of embryonic development (E14.5. The embryos showed peripheral edema, and abnormal heart development as indicated by thinner ventricular wall and reduced cell density at the compact and trabecular areas of myocardium. This is associated with reduced level of proliferating cell nuclear antigen and increased caspase-3 in these tissues. Cardiomyocytes from E14.5 NFAT5(-/- embryos showed a significant reduction of beating rate and abnormal Ca(2+ signaling profile as a consequence of reduced sarco(endoplasmic reticulum Ca(2+-ATPase (SERCA and ryanodine receptor (RyR expressions. Expression of NFAT5 target genes, such as HSP 70 and SMIT were reduced in NFAT5(-/- cardiomyocytes. Our findings demonstrated an essential role of NFAT5 in cardiac development and Ca(2+ signaling. Cardiac failure is most likely responsible for the peripheral edema and death of NFAT5(-/- embryos at E14.5 days.

  20. The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

    Directory of Open Access Journals (Sweden)

    Christian Bleux

    2010-01-01

    Full Text Available Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH which activates specific receptors (GnRHR present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to

  1. A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses

    OpenAIRE

    Juan García-Arriaza; José Luis Nájera; Carmen E Gómez; Nolawit Tewabe; Sorzano, Carlos Oscar S.; Thierry Calandra; Thierry Roger; Mariano Esteban

    2011-01-01

    The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-...

  2. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie

    2006-05-01

    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

  3. Interactions of Neuropathogenic Escherichia coli K1 (RS218 and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Farzana Abubakar Yousuf

    2014-01-01

    Full Text Available Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin, adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (IbeA, CNF1, metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (CNF1, metabolism (D-serine catabolism abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

  4. Lack of organ specific commitment of vagal neural crest cell derivatives as shown by back-transplantation of GFP chicken tissues.

    Science.gov (United States)

    Freem, Lucy J; Delalande, Jean Marie; Campbell, Alison M; Thapar, Nikhil; Burns, Alan J

    2012-01-01

    Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.

  5. Lack of a Dose-Effect Relationship for Pulmonary Function Changes After Stereotactic Body Radiation Therapy for Early-Stage Non-Small Cell Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Guckenberger, Matthias, E-mail: Guckenberger_M@klinik.uni-wuerzburg.de [Department of Radiation Oncology, University Hospital Wuerzburg, Wuerzburg (Germany); Klement, Rainer J. [Department of Radiation Oncology, University Hospital Wuerzburg, Wuerzburg (Germany); Kestin, Larry L. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Hope, Andrew J. [Princess Margaret Hospital, University of Toronto, Toronto, ON (Canada); Belderbos, Jose [The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital, Amsterdam (Netherlands); Werner-Wasik, Maria [Thomas Jefferson University Hospital, Philadelphia, Pennsylvania (United States); Yan, Di [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Sonke, Jan-Jakob [The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital, Amsterdam (Netherlands); Bissonnette, Jean-Pierre [Princess Margaret Hospital, University of Toronto, Toronto, ON (Canada); Xiao, Ying [Thomas Jefferson University Hospital, Philadelphia, Pennsylvania (United States); Grills, Inga S. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States)

    2013-03-15

    Purpose: To evaluate the influence of tumor size, prescription dose, and dose to the lungs on posttreatment pulmonary function test (PFT) changes after stereotactic body radiation therapy (SBRT) for early-stage non-small cell lung cancer (NSCLC). Methods and Materials: The analysis is based on 191 patients treated at 5 international institutions: inclusion criteria were availability of pre- and post-SBRT PFTs and dose-volume histograms of the lung and planning target volume (PTV); patients treated with more than 1 SBRT course were excluded. Correlation between early (1-6 months, median 3 months) and late (7-24 months, median 12 months) PFT changes and tumor size, planning target volume (PTV) dose, and lung doses was assessed using linear regression analysis, receiver operating characteristics analysis, and Lyman's normal tissue complication probability model. The PTV doses were converted to biologically effective doses and lung doses to 2 Gy equivalent doses before correlation analyses. Results: Up to 6 months after SBRT, forced expiratory volume in 1 second and carbon monoxide diffusion capacity changed by −1.4% (95% confidence interval [CI], −3.4% to 0) and −7.6% (95% CI, −10.2% to −3.4%) compared with pretreatment values, respectively. A modest decrease in PFTs was observed 7-24 months after SBRT, with changes of −8.1% (95% CI, −13.3% to −5.3%) and −12.4% (95% CI, −15.5% to −6.9%), respectively. Using linear regression analysis, receiver operating characteristic analysis, and normal tissue complication probability modeling, all evaluated parameters of tumor size, PTV dose, mean lung dose, and absolute and relative volumes of the lung exposed to minimum doses of 5-70 Gy were not correlated with early and late PFT changes. Subgroup analysis based on pre-SBRT PFTs (greater or equal and less than median) did not identify any dose-effect relationship. Conclusions: This study failed to demonstrate a significant dose-effect relationship for

  6. Responsiveness of cytogenetically discrete human myeloma cell lines to lenalidomide: lack of correlation with cereblon and interferon regulatory factor 4 expression levels.

    Science.gov (United States)

    Greenberg, Alexandra J; Walters, Denise K; Kumar, Shaji K; Vincent Rajkumar, S; Jelinek, Diane F

    2013-12-01

    The introduction of novel immunomodulatory drugs (IMiDs) has dramatically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). We examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its downstream target interferon regulatory factor-4 (IRF4), which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that lenalidomide response did not correlate with specific cytogenetic translocations. There were distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μm vs. 0.005 μm, P 0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN and IRF4 expression. Our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. While CRBN and IRF4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment.

  7. Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Markus D. Schofer

    2009-01-01

    Full Text Available Growth factors like bone morphogenetic protein 2 (BMP-2 and vascular endothelial growth factor (VEGF play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid (PLLA nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers.

  8. Lacking power impairs executive functions

    NARCIS (Netherlands)

    Smith, P.K.; Jostmann, N.B.; Galinsky, A.D.; Dijk, W.W. van

    2008-01-01

    Four experiments explored whether lacking power impairs executive functioning, testing the hypothesis that the cognitive presses of powerlessness increase vulnerability to performance decrements during complex executive tasks. In the first three experiments, low power impaired performance on executi

  9. Lack of Fas/CD95 surface expression in highly proliferative leukemic cell lines correlates with loss of CtBP/BARS and redirection of the protein toward giant lysosomal structures.

    Science.gov (United States)

    Monleón, Inmaculada; Iturralde, María; Martínez-Lorenzo, María José; Monteagudo, Luis; Lasierra, Pilar; Larrad, Luis; Piñeiro, Andrés; Naval, Javier; Alava, María Angeles; Anel, Alberto

    2002-07-01

    Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

  10. Lack of cross-protection against invasive pneumonia caused by heterologous strains following murine Streptococcus pneumoniae nasopharyngeal colonisation despite whole cell ELISAs showing significant cross-reactive IgG.

    Science.gov (United States)

    Cohen, Jonathan M; Wilson, Robert; Shah, Pranali; Baxendale, Helen E; Brown, Jeremy S

    2013-05-01

    Prior exposure to intact Streptococcus pneumoniae can induce a protective antibody response to proteins antigens, which prevents subsequent invasive disease. This may be achieved either by colonisation with live bacteria or by immunisation with killed cells. Such approaches could provide novel vaccine strategies that overcome the serotype restriction of conjugate vaccines, and would aim to prevent disease caused by all strains of S. pneumoniae. Serum antibody is required to prevent invasive disease, but which in vitro measure of antibody response correlates best with protective immunity has not been established for protein antigens. Using a model of homologous protection induced through D39 colonisation of CD1 mice, we investigate the potential for heterologous protection against two distinct serotype strains and its serological correlates. Serum IgG from colonised mice bound to heterologous strains in whole cell ELISA at titres similar to the homologous D39. However, no cross-protection was observed, correlating with lack of surface binding of IgG to whole bacteria as measured by flow cytometry. Serum antibody binding to pre-lysed and untreated bacteria in the whole cell ELISA was similar suggesting that ELISA does not discriminate between surface and subcapsular antigens, unlike the flow cytometric approach. Thus, flow cytometric binding to whole bacteria maybe a more reliable correlate of cross-protection for novel species-wide vaccines than whole cell ELISA.

  11. CD4⁺ T-cells, γδ T-cells and B-cells are associated with lack of vaccine protection in Mycobacterium avium subspecies paratuberculosis infection.

    Science.gov (United States)

    de Silva, Kumudika; M Plain, Karren; J Begg, Douglas; C Purdie, Auriol; J Whittington, Richard

    2015-01-01

    Vaccination is one of the strategies used to control the spread of Mycobacterium avium subspecies paratuberculosis (MAP) infection in livestock. Gudair(®) is a widely-used vaccine in sheep and goats and is the only vaccine approved for use in sheep in Australia and New Zealand. This vaccine reduces mortality due to MAP-infection by up to 90% but some sheep remain infectious by shedding MAP in faeces, despite vaccination. In this study, using an experimental infection model in sheep, our aim was to assess differences in immune parameters between vaccinated MAP-exposed sheep in which the vaccine was effective compared to those in which it failed to protect against infection. We assessed immune parameters such as MAP-specific IFNγ, IL-10 and lymphocyte proliferative responses and serum antibody levels. At the end of the trial, 72% of non-vaccinated sheep and 24% of vaccinated sheep were infected, as defined by the detection of viable MAP in intestinal tissues when the trial was terminated at 49 weeks post exposure. There were significant differences in the proliferation of CD4(+), B and γδ T-cells over time in vaccinated sheep in which the vaccine failed to protect against infection compared to the non-infected vaccinated sheep. There were no significant differences in the IFNγ response or serum antibody levels between the vaccinated infected and vaccinated non-infected sheep. These results emphasise the importance of specific lymphocyte subsets in protecting against MAP-infection, especially in vaccinated sheep, and that immune parameters other than the commonly used IFNγ and antibody tests are required when assessing vaccine efficacy.

  12. The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size.

    Science.gov (United States)

    Forrest-Owen, W; Willars, G B; Nahorski, S R; Assefa, D; Davidson, J S; Hislop, J; McArdle, C A

    1999-01-25

    shared PtdIns(4,5)P2 pool. Partial depletion of this pool (GnRH pre-treatment in medium with LiCl) reduced the magnitude of the [3H]IP(X) and Ins(1,4,5)P3 responses to methacholine and GnRH, without altering their temporal profiles. Thus the GnRH receptor does not undergo rapid homologous desensitisation in alphaT3-1 cells in spite of the fact that they can desensitise other endogenous (and recombinant) PLC-activating GPCRs, and the lack of desensitisation cannot be attributed to the existence of GnRH receptor reserve or access to an atypically large or rapidly re-cycled PtdIns(4,5)P2 pool. This unique functional characteristic (mammalian GnRH receptors are the only PLC-activating GPCRs known not to rapidly desensitise) almost certainly therefore reflects the atypical structure of these receptors (mammalian GnRH receptors are the only PLC-activating GPCRs known to lack C-terminal tails).

  13. Lack of cross-resistance to FF-10501, an inhibitor of inosine-5'-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell lines.

    Science.gov (United States)

    Murase, Motohiko; Iwamura, Hiroyuki; Komatsu, Kensuke; Saito, Motoki; Maekawa, Toshihiko; Nakamura, Takaaki; Yokokawa, Takuya; Shimada, Yasuhiro

    2016-02-01

    Resistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine-cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.

  14. Mice lacking neurofibromin develop gastric hyperplasia

    Science.gov (United States)

    Lin, Lu; Chen, Jian; Richardson, James A.

    2009-01-01

    Gastrointestinal (GI) neoplasms are among many manifestations of the genetic disease neurofibromatosis type 1 (NF1). However, the physiological and pathological functions of the Nf1 gene in the GI system have not been fully studied, possibly because of a lack of mouse models. In this study, we generated conditional knockout mice with Nf1 deficiency in the GI tract. These mice develop gastric epithelial hyperplasia and inflammation together with increased cell proliferation and apoptosis. The gastric phenotypes observed in these mutant mice seem to be the consequence of loss of Nf1 in gastric fibroblasts, resulting in paracrine hyperactivation of the ERK pathway in the gastric epithelium. These mice provide a useful model to study the pathogenesis of GI lesions in a subset of patients with NF1 and to investigate the role of the Nf1 gene in the development of GI neoplasms. PMID:19661150

  15. Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1

    DEFF Research Database (Denmark)

    Ugleholdt, Randi; Zhu, Xiaorong; Deacon, Carolyn F;

    2003-01-01

    The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature gluca...

  16. Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11)

    NARCIS (Netherlands)

    J. Neudenberger (J.); M. Hotfilder (Marc); A. Rosemann (Annegret); C. Langebrake (C.); D. Reinhardt (Dirk); R. Pieters (Rob); A. Schrauder (André); M. Schrappe (Martin); S. Röttgers (Silja); J. Harbott (Jochen); J. Vormoor (Josef)

    2006-01-01

    textabstractIt has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for mini

  17. Unresolved questions from the analysis of mice lacking MCU expression.

    Science.gov (United States)

    Murphy, Elizabeth; Pan, Xin; Nguyen, Tiffany; Liu, Jie; Holmström, Kira M; Finkel, Toren

    2014-07-11

    Entry of mitochondrial calcium is believed to play an essential role in regulating bioenergetics and initiating cell death pathways. We have recently described a mouse model lacking MCU expression. Surprisingly, these mice are viable and the cells and tissues from these animals do not exhibit any marked protection from cell death. Here, we discuss our findings as well as potential explanations for some of the more unexpected results.

  18. Development of ADA Against Recombinant Human Interferon Beta in Immune Tolerant Mice Requires Rapid Recruitment of CD4(+) T Cells, Induces Formation of Germinal Centers but Lacks Susceptibility for (Most) Adjuvants

    NARCIS (Netherlands)

    Kijanka, Grzegorz; Sauerborn, Melody; Boon, Louis; Schellekens, Huub; Brinks, Vera

    2015-01-01

    Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent (invo

  19. Lack of RNase L attenuates macrophage functions.

    Directory of Open Access Journals (Sweden)

    Xin Yi

    Full Text Available Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α, interleukin (IL-1β, IL-6, and cyclooxygenase-2 (Cox-2 by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.Bone marrow-derived macrophages (BMMs were generated from RNase L(+/+and (-/- mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2. Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

  20. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor.

    Science.gov (United States)

    Andrews, P W; Damjanov, I; Simon, D; Dignazio, M

    1985-01-01

    Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.

  1. Lack of prognostic significance of BCL2 and p53 protein overexpression in elderly patients with diffuse large B-cell non-Hodgkin's lymphoma : Results from a population-based non-Hodgkin's lymphoma registry

    NARCIS (Netherlands)

    Maartense, E; Kramer, MHH; Le Cessie, S; Kluin-Nelemans, JC; Kluin, PM; Snijder, S; Noordijk, EM

    2004-01-01

    The prognostic significance of age was studied in 372 patients with diffuse large B-cell non-Hodgkin's lymphoma, in relation to the prognostic factors of overexpressed BCL2 and p53 oncoprotein. Overexpression of BCL2 and p53 oncoprotein was defined when more than 50% of the tumor cells showed positi

  2. Multiple defects in Escherichia coli mutants lacking HU protein.

    OpenAIRE

    Huisman, O; Faelen, M; Girard, D; Jaffé, A; Toussaint, A; Rouvière-Yaniv, J

    1989-01-01

    The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize,...

  3. Lack of Set Theory Relevant Prerequisite Knowledge

    Science.gov (United States)

    Dogan-Dunlap, Hamide

    2006-01-01

    Many students struggle with college mathematics topics due to a lack of mastery of prerequisite knowledge. Set theory language is one such prerequisite for linear algebra courses. Many students' mistakes on linear algebra questions reveal a lack of mastery of set theory knowledge. This paper reports the findings of a qualitative analysis of a…

  4. Androgen receptor (AR) pathophysiological roles in androgen-related diseases in skin, bone/muscle, metabolic syndrome and neuron/immune systems: lessons learned from mice lacking AR in specific cells.

    Science.gov (United States)

    Chang, Chawnshang; Yeh, Shuyuan; Lee, Soo Ok; Chang, Ta-Min

    2013-01-01

    The androgen receptor (AR) is expressed ubiquitously and plays a variety of roles in a vast number of physiological and pathophysiological processes. Recent studies of AR knockout (ARKO) mouse models, particularly the cell type- or tissue-specific ARKO models, have uncovered many AR cell type- or tissue-specific pathophysiological roles in mice, which otherwise would not be delineated from conventional castration and androgen insensitivity syndrome studies. Thus, the AR in various specific cell types plays pivotal roles in production and maturation of immune cells, bone mineralization, and muscle growth. In metabolism, the ARs in brain, particularly in the hypothalamus, and the liver appear to participate in regulation of insulin sensitivity and glucose homeostasis. The AR also plays key roles in cutaneous wound healing and cardiovascular diseases, including atherosclerosis and abdominal aortic aneurysm. This article will discuss the results obtained from the total, cell type-, or tissue-specific ARKO models. The understanding of AR cell type- or tissue-specific physiological and pathophysiological roles using these in vivo mouse models will provide useful information in uncovering AR roles in humans and eventually help us to develop better therapies via targeting the AR or its downstream signaling molecules to combat androgen/AR-related diseases.

  5. Lack of Exercise Might Invite Dementia

    Science.gov (United States)

    ... fullstory_163264.html Lack of Exercise Might Invite Dementia Study found being sedentary may make you as ... TV may make you as likely to develop dementia as people genetically predisposed to the condition, a ...

  6. LACK - a VoIP Steganographic Method

    CERN Document Server

    Mazurczyk, Wojciech

    2008-01-01

    The paper presents a new steganographic method called LACK (Lost Audio PaCKets Steganography) which is intended mainly for VoIP. The method is presented in a broader context of network steganography and of VoIP steganography in particular. The analytical results presented in the paper concern the influence of LACK's hidden data insertion procedure on the method's impact on quality of voice transmission and its resistance to steganalysis.

  7. Major role for a 3p21 region and lack of involvement of the t(3;8) breakpoint region in the development of renal cell carcinoma suggested by loss of heterozygosity analysis

    NARCIS (Netherlands)

    van den Berg, Anke; Hulsbeek, MMF; deJong, D; Kok, K; Veldhuis, PMJF; Roche, J; Buys, CHCM

    1996-01-01

    In a loss of heterozygosity analysis of 3p, we examined 44 sporadic cases of renal cell carcinoma (RCC) and matched normal tissue with 18 markers distributed over the whole p-arm. The majority of these markers clustered in three regions that have been suggested to be involved in the development of R

  8. Abundance of DLK1, differential expression of CYP11B1, CYP21A2 and MC2R, and lack of INSL3 distinguish testicular adrenal rest tumours from Leydig cell tumours

    DEFF Research Database (Denmark)

    Lottrup, Grete; Nielsen, John Erik; Skakkebæk, Niels Erik;

    2015-01-01

    in the differential diagnosis of TARTs and malignant LC tumours (LCTs). METHODS: We investigated mRNA and protein expression of testicular steroidogenic enzymes; CYP11A1 and HSD3B1/2, markers of adrenal steroidogenesis; CYP11B1, CYP21A2 and ACTH receptor/melanocortin 2 receptor (MC2R), and markers of LC maturation...... profiles in TART and fetal adrenals as well as the presence of classical markers of adrenal steroidogenesis lend support to the hypothesis that TART develops from a displaced adrenal cell type. Malignant LCTs seem to have lost DLK1 expression and do not resemble immature LCs. The different expression...

  9. Lack of prognostic and predictive value of CA IX in radiotherapy of squamous cell carcinoma of the head and neck with known modifiable hypoxia: An evaluation of the DAHANCA 5 study

    DEFF Research Database (Denmark)

    Eriksen, Jesper Grau; Overgaard, Jens

    2007-01-01

    BACKGROUND AND PURPOSE: CA IX is suggested to be an endogenous marker of hypoxia in tumours like squamous cell carcinomas of the head and neck (HNSCC). The aim of the present study was to investigate whether CA IX served as a prognostic factor for outcome in a large population of HNSCC and if CA IX......+/-the hypoxic radiosensitizer nimorazole. CA IX was measured using immunohistochemistry and results were divided into four groups of CA IX expression: 30% of the tumour area with positive membrane staining. Locoregional control and disease-specific survival were used as endpoints...

  10. Myxoma virus lacking the pyrin-like protein M013 is sensed in human myeloid cells by both NLRP3 and multiple Toll-like receptors, which independently activate the inflammasome and NF-κB innate response pathways.

    Science.gov (United States)

    Rahman, Masmudur M; McFadden, Grant

    2011-12-01

    The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1β (IL-1β), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1β are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1β in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1β was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1β in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends

  11. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro.

    Science.gov (United States)

    Son, D S; Ushinohama, K; Gao, X; Taylor, C C; Roby, K F; Rozman, K K; Terranova, P F

    1999-01-01

    The main purpose of this study was to investigate the direct effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHR) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GC), theca-interstitial cells (TIC), and whole ovarian dispersates derived from the ovary of IHR were investigated. In the ovulation model, rats were hypophysectomized on Day 23 of age. On Day 26, the IHR were given 20 microg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicular development. Fifty-two hours after eCG, 10 IU hCG was given to induce ovulation. TCDD (20 microg/kg) blocked ovulation and reduced ovarian weight in IHR. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 h after eCG. except for a two-fold increase in ovarian concentration of A4 at 48 h after TCDD. However, this higher concentration of A4 at 48 h after TCDD did not reflect that of A4 in sera and did not correlate with E2 in either sera or ovaries. In isolated GC from untreated IHR, TCDD (0.1 to 100 nM) had no significant effect on P4 and E2 after stimulation by LH or FSH. In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1 to 800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using RT-PCR, AhR mRNA was shown to be expressed constitutively in the whole ovary of IHR with maximum down-regulation at 6 h after TCDD (20 microg/kg). Ovarian CYP1A1 was induced maximally at 6 h after TCDD, whereas CYP1B1 could not be detected. The induction of AhR related genes by TCDD in the ovary implies the existence of AhR-mediated signal

  12. Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-{kappa}B-STAT3-directed gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu; Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

    2011-07-01

    Mitochondrial DNA depleted ({rho}{sup 0}) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-{kappa}B and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental {rho}{sup +} HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in {rho}{sup 0} cells compared to {rho}{sup +} HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, {Oota}L17{Beta}, {Oota}L18, {Oota}L19, and {Oota}L28{Beta}) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-{kappa}B and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-{kappa}B/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in {rho}{sup +} HSF, but this response was substantially decreased in {rho}{sup 0} HSF. Suppression of the IKK-NF-{kappa}B pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated {rho}{sup +} HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-{kappa}B activation was partially lost in {rho}{sup 0} HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-{kappa}B targets, further suppressing IL6

  13. Lack of survival advantage in patients with advanced, resectable squamous cell carcinoma of the oral cavity receiving induction chemotherapy with cisplatin (CDDP), docetaxel (TXT) and 5-fluorouracil (5FU).

    Science.gov (United States)

    Umeda, Masahiro; Komatsubara, Hideki; Ojima, Yasutaka; Minamikawa, Tsutomu; Shigeta, Takashi; Shibuya, Yasuyuki; Yokoo, Satoshi; Komori, Takahide

    2004-01-01

    Cisplatin-based neoadjuvant chemotherapy (NAC) has been reported to increase survival of patients with nasopharyngeal carcinoma, and organ preservation in those with laryngeal carcinoma, but its efficacy for other head and neck carcinomas is still controversial. We examined the effects of NAC for patients with stage III-IV squamous cell carcinoma of the oral cavity. The patients were divided into two groups; 9 patients who underwent NAC consisting of one course of cisplatin (CDDP), docetaxel (TXT) and 5-fluorouracil (5FU) followed by surgery (NAC group), and 18 patients who underwent surgery alone (control group). Complete response (CR) was not observed, but partial response (PR) was obtained in 6 of 9 patients (33%) of the NAC group. The 3-year survival rate was 29.6% in the NAC group and 81.5% in the control group. Although any valid conclusions could not be drawn because of the small number of patients examined here, NAC with CDDP, TXT and 5FU offered no advantages over standard treatment for advanced oral cancer in terms of survival.

  14. Mucosal alpha-papillomaviruses are not associated with esophageal squamous cell carcinomas: Lack of mechanistic evidence from South Africa, China and Iran and from a world-wide meta-analysis.

    Science.gov (United States)

    Halec, Gordana; Schmitt, Markus; Egger, Sam; Abnet, Christian C; Babb, Chantal; Dawsey, Sanford M; Flechtenmacher, Christa; Gheit, Tarik; Hale, Martin; Holzinger, Dana; Malekzadeh, Reza; Taylor, Philip R; Tommasino, Massimo; Urban, Margaret I; Waterboer, Tim; Pawlita, Michael; Sitas, Freddy

    2016-07-01

    Epidemiological and mechanistic evidence on the causative role of human papillomaviruses (HPV) in esophageal squamous cell carcinoma (ESCC) is unclear. We retrieved alcohol- and formalin-fixed paraffin-embedded ESCC tissues from 133 patients seropositive for antibodies against HPV early proteins, from high-incidence ESCC regions: South Africa, China and Iran. With rigorous care to prevent nucleic acid contamination, we analyzed these tissues for the presence of 51 mucosotropic human alpha-papillomaviruses by two sensitive, broad-spectrum genotyping methods, and for the markers of HPV-transformed phenotype: (i) HPV16/18 viral loads by quantitative real-time PCR, (ii) type-specific viral mRNA by E6*I/E6 full-length RT-PCR assays and (iii) expression of cellular protein p16(INK4a). Of 118 analyzable ESCC tissues, 10 (8%) were positive for DNA of HPV types: 16 (4 tumors); 33, 35, 45 (1 tumor each); 11 (2 tumors) and 16, 70 double infection (1 tumor). Inconsistent HPV DNA+ findings by two genotyping methods and negativity in qPCR indicated very low viral loads. A single HPV16 DNA+ tumor additionally harbored HPV16 E6*I mRNA but was p16(INK4a) negative (HPV16 E1 seropositive patient). Another HPV16 DNA+ tumor from an HPV16 E6 seropositive patient showed p16(INK4a) upregulation but no HPV16 mRNA. In the tumor tissues of these serologically preselected ESCC patients, we did not find consistent presence of HPV DNA, HPV mRNA or p16(INK4a) upregulation. These results were supported by a meta-analysis of 14 other similar studies regarding HPV-transformation of ESCC. Our study does not support the etiological role of the 51 analyzed mucosotropic HPV types in the ESCC carcinogenesis.

  15. Mice Lacking Hbp1 Function Are Viable and Fertile

    Science.gov (United States)

    Wilhelm, Dagmar; Jans, David A.; Bowles, Josephine; Koopman, Peter

    2017-01-01

    Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. PMID:28107452

  16. Conceptualising the lack of health insurance coverage.

    Science.gov (United States)

    Davis, J B

    2000-01-01

    This paper examines the lack of health insurance coverage in the US as a public policy issue. It first compares the problem of health insurance coverage to the problem of unemployment to show that in terms of the numbers of individuals affected lack of health insurance is a problem comparable in importance to the problem of unemployment. Secondly, the paper discusses the methodology involved in measuring health insurance coverage, and argues that the current method of estimation of the uninsured underestimates the extent that individuals go without health insurance. Third, the paper briefly introduces Amartya Sen's functioning and capabilities framework to suggest a way of representing the extent to which individuals are uninsured. Fourth, the paper sketches a means of operationalizing the Sen representation of the uninsured in terms of the disability-adjusted life year (DALY) measure.

  17. Reduced alcohol consumption in mice lacking preprodynorphin.

    OpenAIRE

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

    2006-01-01

    Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower a...

  18. Why does Colombia lack agricultural commodity futures?

    Directory of Open Access Journals (Sweden)

    Pablo Moreno-Alemay

    2015-11-01

    Full Text Available This article explores the reasons why futures contracts are not traded as an alternative to price hedging for agricultural goods in Colombia. Based on surveys, interviews and statistical analysis, this study identified that conceptual gaps in contract negotiation, lack of consensus in the agricultural sector regarding the use of financial mechanisms and the sector’s infrequent contact with Colombia’s financial institutions, are the main reasons why a futures contracts market has not emerged.

  19. Quantum preparation uncertainty and lack of information

    Science.gov (United States)

    Rozpędek, Filip; Kaniewski, Jędrzej; Coles, Patrick J.; Wehner, Stephanie

    2017-02-01

    The quantum uncertainty principle famously predicts that there exist measurements that are inherently incompatible, in the sense that their outcomes cannot be predicted simultaneously. In contrast, no such uncertainty exists in the classical domain, where all uncertainty results from ignorance about the exact state of the physical system. Here, we critically examine the concept of preparation uncertainty and ask whether similarly in the quantum regime, some of the uncertainty that we observe can actually also be understood as a lack of information (LOI), albeit a lack of quantum information. We answer this question affirmatively by showing that for the well known measurements employed in BB84 quantum key distribution (Bennett and Brassard 1984 Int. Conf. on Computer System and Signal Processing), the amount of uncertainty can indeed be related to the amount of available information about additional registers determining the choice of the measurement. We proceed to show that also for other measurements the amount of uncertainty is in part connected to a LOI. Finally, we discuss the conceptual implications of our observation to the security of cryptographic protocols that make use of BB84 states.

  20. Lack of transplacental transmission of Bartonella bovis.

    Science.gov (United States)

    Chastant-Maillard, S; Boulouis, H-J; Reynaud, K; Thoumire, S; Gandoin, C; Bouillin, C; Cordonnier, N; Maillard, R

    2015-02-01

    Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

  1. Energy brands lack vitality; Energiemerken ontberen vitaliteit

    Energy Technology Data Exchange (ETDEWEB)

    Godri, S.; Wilders, E. [CONSULT Brand Strategy, Y and R Advertising, Amsterdam (Netherlands)

    2004-06-01

    The three Dutch energy companies (Nuon, Essent and Eneco Energie) have relatively little brand strength. The brands are not perceived to be sufficiently different from one another and are not valued by consumers. With liberalisation imminent, this is hardly a strong starting point. How can you win over consumers if it is not clear what is on offer? In the business market, decision-makers are better placed to distinguish between brands. However, the brands lack vitality in this sector of the market too. The only consolation is that the situation is by no means exclusive to the Netherlands. [Dutch] De merksterkte van de drie Nederlandse energiebedrijven Nuon, Essent en Eneco Energie, is gering. De energiemerken onderscheiden zich niet genoeg en missen waardering. Met het oog op de aanstaande liberalisering geen goede uitgangspositie. Hoe kun je consumenten voor je winnen als onduidelijk is waarvoor ze moeten kiezen? Op de zakelijk markt zijn de beslissers beter in staat om de merken te onderscheiden. Maar ook hier missen de energiemerken vitaliteit. Een troost: Nederlandse energiebedrijven zijn niet de enige met dit probleem.

  2. Lack of universal scaling in magnetohydrodynamic turbulence

    Science.gov (United States)

    Pouquet, A.; Brachet, M.; Krstulovic, G.; Lee, E.; Mininni, P.; Rosenberg, D. L.

    2012-12-01

    Universality is often viewed as a hallmark of turbulent flows, with a search for scaling exponents that derive from intrinsic dynamics and do not depend on initial conditions or forcing, the Kolmogorov law for the energy spectrum of an incompressible fluid being the best known case. However, in the presence of waves due to an external agent such as rotation, stratification or a strong large-scale magnetic field B0, different regimes -- such as weak or strong turbulence, may arise and thus, different scaling behavior may arise as well. This is observed for example in the ocean, and it leads to different mixing and transport properties. In this talk, we shall first review, in the context of MHD turbulence, the phenomenological models that can be constructed using the following plausible dimensionless parameters: (i) RT, the ratio of characteristic time scales (here, the wave period Tw=L_0/B_0 and the eddy-turn-over time based on large-scale length and velocity, TNL=L0/U_0; (ii) RE, the ratio of magnetic to kinetic energy EM/E_V; and (iii) RA, the degree of alignment between the velocity and the magnetic field \\cos(v,b), or between the magnetic potential and magnetic induction, \\cos(A,b). Note that these ratios can also be defined at scale ℓ of velocity uℓ (as opposed to L0, U0), and thus one can consider as well the variation of such ratios across scales. We shall then contrast these models with data stemming from (mostly) solar observations that indicate a clear lack of universal scaling behavior. Similarly, a number of direct numerical simulations (DNS) including some at high resolution, in the spin-down of forcing case, in the presence of boundaries or not, and with or without an imposed strong external magnetic field B0, all point-out to different energy spectra, although the attainable Reynolds numbers in present-day DNS are still limited when contrasted with geophysical and astrophysical flows. In particular, we shall show that, when using as initial

  3. Lack of efficacy of ergocalciferol repletion

    Directory of Open Access Journals (Sweden)

    Thomas Wasser

    2012-04-01

    Full Text Available Introduction: Vitamin D has become an area of intensive scrutiny, both in medical and lay literature. However, there are limited data to suggest proper repletion regimens for those patients who have hypovitaminosis D. Consequently, various methods are used in clinical practice. The aim of this study was to assess the efficacy of various treatment strategies for hypovitaminosis D in an ambulatory internal medicine practice. Methods: A retrospective chart review between October 2005 and June 2010 of a suburban internal medicine practice was performed via query of the electronic medical record (Centricity, General Electric Healthcare, UK. Patients with a 25-hydroxyvitamin D concentration less than 32 mg/dl were identified and treated. Treatment success was defined as 25-hydroxyvitamin D concentrations greater than 32 mg/dl. Statistical analysis to assess changes in vitamin D level controlling for season, comorbidities, and demographics were used. Results: A total of 607 treatment episodes were identified, with 395 excluded due to lack of follow-up vitamin D level within 16 weeks, no treatment documented, topical treatment, doxercalciferol treatment, or non-compliance. Of the remaining patients, there were 212 treatment instances on 178 patients. Ergocalciferol 50,000 international units (IU was used most frequently (71.4% of the time.. A higher initial vitamin D level was positively associated with treatment success (adjusted odds ratio = 1.11, p=0.002. Increased doses of ergocalciferol increased the likelihood of treatment success (p=0.0011. Seasonal variation was related to posttreatment 25-hydroxyvitamin D concentration as was body mass index (BMI (p=0.003 and p=0.044. Conclusion: Pretreatment levels of 25-hydroxyvitamin D, BMI, season, and vitamin D dose are predictors of successful hypovitaminosis D treatment. Our data suggest that patients with initial 25-hydroxyvitamin D concentrations of <20 should be treated with a higher total dose of

  4. Thyroid development in zebrafish lacking Taz.

    Science.gov (United States)

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, Stephan; Zannini, Mariastella; Sordino, Paolo

    2015-11-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated knockdown of wwtr1, the gene encoding zebrafish Taz. The wwtr1 gene is expressed in the thyroid primordium and pharyngeal tissue of developing zebrafish. Compared to mammalian cells, in which Taz promotes expression of thyroid transcription factors and thyroid differentiation genes, wwtr1 MO injection in zebrafish had little or no effect on the expression of thyroid transcription factors, and differentially altered the expression of thyroid differentiation genes. Analysis of wwtr1 morphants at later stages of development revealed that the number and the lumen of thyroid follicles, and the number of thyroid follicle cells, were significantly smaller. In addition, Taz-depleted larvae displayed patterning defects in ventral cranial vessels that correlate with lateral displacement of thyroid follicles. These findings indicate that the zebrafish Taz protein is needed for the normal differentiation of the thyroid and are the first to suggest that Taz confers growth advantage to the endocrine gland.

  5. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  6. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.

    Science.gov (United States)

    Pan, Xin; Liu, Jie; Nguyen, Tiffany; Liu, Chengyu; Sun, Junhui; Teng, Yanjie; Fergusson, Maria M; Rovira, Ilsa I; Allen, Michele; Springer, Danielle A; Aponte, Angel M; Gucek, Marjan; Balaban, Robert S; Murphy, Elizabeth; Finkel, Toren

    2013-12-01

    Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death, although MCU(-/-) hearts fail to respond to the PTP inhibitor cyclosporin A. Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.

  7. Indigenous Traditional Medical Practitioners’ Lack of Formal Medical Education Impacts their Choices of Information Resources for the Treatment of Sickle Cell Anemia. A Review of: Olatokun, W. M., & Ajagbe, E. (2010. Analyzing traditional medical practitioners’ information-seeking behavior using Taylor’s information-use environment model. Journal of Librarianship and Information Science, 42, 122-135.

    Directory of Open Access Journals (Sweden)

    Maria C. Melssen

    2011-06-01

    sources. The informal sources most commonly used are local associations (55%, colleagues (55%, and master healers (52.5%. Such formal resources as medical journals, seminars or workshops, the Internet, and libraries are rarely if ever used. The factors influencing the practitioners’ resource choice include relevance (87.8%, suitability (70%, and availability (67.5%.Many practitioners also refer their patients to other traditional medical practitioners; however, very few (27.5% refer patients to orthodox physicians. The traditional practitioners felt that they can treat their patients on their own and do not need the orthodox physician’s help. The traditional practitioners also feel that there is little or no information sharing between the traditional practitioners and the orthodox physicians: the only time information is exchanged between the two groups is when the orthodox physicians want to conduct research on traditional medical practices.Conclusion – The traditional practitioners rely heavily on information from local experts to guide their treatment plans for sickle cell anemia patients. The success or failure of a given treatment plan is also based on what did or did not work in the past. These practitioners do not have a formal education and have a low literacy level. This group is not recognized by western medical culture as a result of their lack of professional, western medical training. Another issue is that there is not a solid documentation system of the treatment and management of sickle cell anemia by this group. This is due to their fears of having their methods “stolen” by fellow practitioners. Recommendations by the authors include having the association leaders document and track the treatment and disease management methods used by their members and implementing a training program for the indigenous traditional medicine practitioners. Further research needed includes exploring the various ways to integrate western medical practices with

  8. New constitutive latex osmotin-like proteins lacking antifungal activity.

    Science.gov (United States)

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  9. Association between friction and wear in diarthrodial joints lacking lubricin

    Science.gov (United States)

    Jay, Gregory D; Torres, Jahn R; Rhee, David K; Helminen, Heikki J; Hytinnen, Mika M; Cha, Chung-Ja; Elsaid, Khaled; Kim, Kyung-Suk; Cui, Yajun; Warman, Matthew L

    2007-01-01

    Objective The glycoprotein lubricin (encoded by the gene Prg4) is secreted by surface chondrocytes and synovial cells, and has been shown to reduce friction in vitro. In contrast to man-made bearings, mammalian diarthrodial joints must endogenously produce friction-reducing agents. This study was undertaken to investigate whether friction is associated with wear. Methods The lubricating ability of synovial fluid (SF) samples from humans with genetic lubricin deficiency was tested in vitro. The coefficient of friction in the knee joints of normal and lubricin-null mice was measured ex vivo; these joints were also studied by light and electron microscopy. Atomic force microscopy was used to image and measure how lubricin reduces friction in vitro. Results SF lacking lubricin failed to reduce friction in the boundary mode. Joints of lubricin-null mice showed early wear and higher friction than joints from their wild-type counterparts. Lubricin self-organized and reduced the work of adhesion between apposing asperities. Conclusion These data show that friction is coupled with wear at the cartilage surface in vivo. They imply that acquired lubricin degradation occurring in inflammatory joint diseases predisposes the cartilage to damage. Lastly, they suggest that lubricin, or similar biomolecules, will have applications in man-made devices in which reducing friction is essential. PMID:17968947

  10. Lack of Galanin 3 Receptor Aggravates Murine Autoimmune Arthritis.

    Science.gov (United States)

    Botz, Bálint; Kemény, Ágnes; Brunner, Susanne M; Locker, Felix; Csepregi, Janka; Mócsai, Attila; Pintér, Erika; McDougall, Jason J; Kofler, Barbara; Helyes, Zsuzsanna

    2016-06-01

    Neurogenic inflammation mediated by peptidergic sensory nerves has a crucial impact on the pathogenesis of various joint diseases. Galanin is a regulatory sensory neuropeptide, which has been shown to attenuate neurogenic inflammation, modulate neutrophil activation, and be involved in the development of adjuvant arthritis, but our current understanding about its targets and physiological importance is incomplete. Among the receptors of galanin (GAL1-3), GAL3 has been found to be the most abundantly expressed in the vasculature and on the surface of some immune cells. However, since there are minimal in vivo data on the role of GAL3 in joint diseases, we analyzed its involvement in different inflammatory mechanisms of the K/BxN serum transfer-model of autoimmune arthritis employing GAL 3 gene-deficient mice. After arthritis induction, GAL3 knockouts demonstrated increased clinical disease severity and earlier hindlimb edema than wild types. Vascular hyperpermeability determined by in vivo fluorescence imaging was also elevated compared to the wild-type controls. However, neutrophil accumulation detected by in vivo luminescence imaging or arthritic mechanical hyperalgesia was not altered by the lack of the GAL3 receptor. Our findings suggest that GAL3 has anti-inflammatory properties in joints by inhibiting vascular hyperpermeability and consequent edema formation.

  11. Characterization of nonconventional hepatitis B viruses lacking the core promoter.

    Science.gov (United States)

    Chang, Shau-Feng; Chang, Shih-Hsuan; Li, Bi-Chen; Will, Hans; Netter, Hans Jürgen

    2004-12-20

    The core gene (C-gene) promoter and regulatory sequences play a central role in the hepatitis B virus (HBV) life cycle. They are essential for the synthesis of the pregenomic and precore mRNA. The pregenomic RNA is the template required for replication and also the template for the synthesis of the core protein and polymerase. Here, we report the in vivo existence and functional characterization of HBV variants that lack the C-gene promoter region and the regulatory sequences located therein. HBV promoter fragments were isolated by PCR from sera of chronic carriers and characterized. Truncated promoter elements were identified, and then tested in the context of wild-type genomes in the HuH-7 cell line. The expression of the recombinant HBV genome resulted in the synthesis of surface proteins, and low level of core protein as well as a transcript pattern similar to, but smaller in size to wild-type virus. The recombinant HBV genome with the truncated promoter region produced pregenomic RNA-like transcripts. These transcripts were encapsidated and reverse transcribed when complemented by sufficient core and polymerase protein. These date provide an explanation as to why such deletion mutants of HBV can be produced at all, they highlight the functional potentials of viral sequences activated by mutations and may be of relevance for viral evolution and persistence.

  12. Why Art Education Lacks Social Relevance: A Contextual Analysis.

    Science.gov (United States)

    Bersson, Robert

    1987-01-01

    This article examines why both the individual-centered and discipline-centered approaches to art education lack social relevance. Maintains that the lack of a social orientation in these two approaches is due to our social context and our capitalistic system. Proposes that democratic principles serve as the basis of a socially relevant,…

  13. 29 CFR 18.602 - Lack of personal knowledge.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Lack of personal knowledge. 18.602 Section 18.602 Labor... OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A... witness has personal knowledge of the matter. Evidence to prove personal knowledge may, but need...

  14. Lack of conventional ATPase properties in CFTR chloride channel gating.

    Science.gov (United States)

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  15. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Directory of Open Access Journals (Sweden)

    Nour Hammoudeh

    2014-12-01

    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  16. Muscle Regeneration and Myogenic Differentiation Defects in Mice Lacking TIS7

    OpenAIRE

    Vadivelu, Santhosh K.; Kurzbauer, Robert; Dieplinger, Benjamin; ZWEYER, MARGIT; Schafer, Ralf; Wernig, Anton; Vietor, Ilja; Huber, Lukas A.

    2004-01-01

    The tetradecanoyl phorbol acetate-induced sequence 7 gene (tis7) is regulated during cell fate processes and functions as a transcriptional coregulator. Here, we describe the generation and analysis of mice lacking the tis7 gene. Surprisingly, TIS7 knockout mice show no gross histological abnormalities and are fertile. Disruption of the tis7 gene by homologous recombination delayed muscle regeneration and altered the isometric contractile properties of skeletal muscles after muscle crush dama...

  17. Special Relativity in Week One: 4) Lack of Simultaneity

    Science.gov (United States)

    Huggins, Elisha

    2011-01-01

    This is our final article on teaching special relativity in the first week of an introductory physics course. One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the…

  18. Garlic exhibits lack of control over gastrointestinal nematodes in goats

    Science.gov (United States)

    Gastrointestinal nematodes (GIN) continue to hinder small ruminant production because of anthelmintic resistance and lack of effective products for GIN control in organic production. The objective of this study was to examine the effectiveness of a commercially available certified organic garlic pr...

  19. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    Science.gov (United States)

    Williams, Margaret M; Sen, Kathryn; Weigand, Michael R; Skoff, Tami H; Cunningham, Victoria A; Halse, Tanya A; Tondella, M Lucia

    2016-02-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

  20. Siim Nestor soovitab : lack of Eoins / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2008-01-01

    Väikefirma Seksound annab sel nädalavahetusel välja Viljandi indiebändi Lack of Eoins esikplaadi "Echo Group" (plaadiesitlused 11. dets. Tallinnas Von Krahlis ja 12. dets. Tartus Genialistide klubis, esinevad ka Ans. Andur ja Popidiot, plaate keerutavad Hannes Praks ja Taavi Laatsit)

  1. Lack of peaceful resolution with Israel: economic cost for Palestinians

    NARCIS (Netherlands)

    P.M.C. de Boer (Paul)

    2008-01-01

    textabstractWe propose to estimate the economic cost for Palestine and for Palestinian residents due to the lack of peaceful resolution with Israel. Thereto we make use of the consensus estimates of the International Monetary Fund (IMF) and the World Bank (WB) of real growth rates of economic variab

  2. Lack of Clarity in University Teaching: A Case Study.

    Science.gov (United States)

    Hativa, Nira

    1998-01-01

    A study used qualitative methods to examine lack of clarity and need for inference in teaching of an undergraduate physics course for nonscience majors. Evidence from several data sources converge, revealing very low levels of student understanding of material presented and strong dissatisfaction with instruction, and also insights into teacher…

  3. Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

    Science.gov (United States)

    Farkas, Jozsef; Sandor, Balazs; Tamas, Andrea; Kiss, Peter; Hashimoto, Hitoshi; Nagy, Andras D; Fulop, Balazs D; Juhasz, Tamas; Manavalan, Sridharan; Reglodi, Dora

    2017-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

  4. Gene expression profiling of gastric mucosa in mice lacking CCK and gastrin receptors

    DEFF Research Database (Denmark)

    Zhao, Chun-Mei; Kodama, Yosuke; Flatberg, Arnar

    2014-01-01

    acid secretion was impaired and the ECL cell signaling pathway was inhibited in CCK2 receptor KO mice but not in CCK1 receptor KO mice. However, in CCK1+2 receptor double KO mice the acid secretion in response to pylorus ligation-induced vagal stimulation and the ECL cell pathway were partially...... hydroxylase probably in trefoil peptide2-immunoreactive cells. In conclusion, mice lacking CCK receptors exhibited a functional shift from the gastrin-CCK pathways to the neuronal pathway in control of the ECL cells and eventually the acid secretion. Taking the present data together with previous findings, we......The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric...

  5. Deriving mechanisms responsible for the lack of correlation between hypoxia and acidity in solid tumors.

    Directory of Open Access Journals (Sweden)

    Hamid R Molavian

    Full Text Available Hypoxia and acidity are two main microenvironmental factors intimately associated with solid tumors and play critical roles in tumor growth and metastasis. The experimental results of Helmlinger and colleagues (Nature Medicine 3, 177, 1997 provide evidence of a lack of correlation between these factors on the micrometer scale in vivo and further show that the distribution of pH and pO(2 are heterogeneous. Here, using computational simulations, grounded in these experimental results, we show that the lack of correlation between pH and pO(2 and the heterogeneity in their shapes are related to the heterogeneous concentration of buffers and oxygen in the blood vessels, further amplified by the network of blood vessels and the cell metabolism. We also demonstrate that, although the judicious administration of anti-angiogenesis agents (normalization process in tumors may lead to recovery of the correlation between hypoxia and acidity, it may not normalize the pH throughout the whole tumor. However, an increase in the buffering capacity inside the blood vessels does appear to increase the extracellular pH throughout the whole tumor. Based on these results, we propose that the application of anti-angiogenic agents and at the same time increasing the buffering capacity of the tumor extracellular environment may be the most efficient way of normalizing the tumor microenvironment. As a by-product of our simulation we show that the recently observed lack of correlation between glucose consumption and hypoxia in cells which rely on respiration is related to the inhomogeneous consumption of glucose to oxygen concentration. We also demonstrate that this lack of correlation in cells which rely on glycolysis could be related to the heterogeneous concentration of oxygen inside the blood vessels.

  6. LACK OF VITAMIN B12 AFTER ROUX Y GASTRIC BYPASS

    OpenAIRE

    Reginaldo, Greise Janaina; Silva, Alice Freitas da

    2014-01-01

    Obesity is a public health issue, with several factors as its cause. It may be related to several co-morbidities such as diabetes mellitus II, systemic arterial pressure, dyslipidemia, cancer and others. Current research verified aspects of bariatric surgery with special focus on alterations caused by the lack of vitamin B12. The bibliographical review evaluated scientific papers, particularly recent ones, to obtain data on the theme. Obesity is defined as a disease characterized by the accum...

  7. Importance Of Business Planning In An Environment Lacking Economic Predictability

    OpenAIRE

    Ciumara Tudor

    2010-01-01

    The importance of business planning in an environment that lacks economic predictability is enhanced because it increases the ability of firms to be proactive. It is however necessary to accept the fundamental assumption that planning activity is conducted in a turbulent environment, where unpredictable events occur frequently. Planning is possible even when changes are common and difficult to predict, by adopting a flexible model to address all significant aspects.

  8. Indigenous Traditional Medical Practitioners’ Lack of Formal Medical Education Impacts their Choices of Information Resources for the Treatment of Sickle Cell Anemia. A Review of: Olatokun, W. M., & Ajagbe, E. (2010). Analyzing traditional medical practitioners’ information-seeking behavior using Taylor’s information-use environment model. Journal of Librarianship and Information Science, 42, 122-135.

    OpenAIRE

    Maria C. Melssen

    2011-01-01

    Objective – To determine the information seeking behaviours of traditional medical practitioners who treat sickle cell anemia patients.Design – Qualitative, interviewer-administered, structured questionnaire.Setting – City and surrounding rural area of Ibadan, Nigeria.Subjects – The researchers selected for this study 160 indigenous traditional medical practitioners who specialize in the treatment of sickle cell anemia. The majority of the subjects were male, with 96 male and 64 female. The p...

  9. REPROBATION AND LACK OF INTEREST IN MECHATRONICS ENGINEERING STUDENTS

    Directory of Open Access Journals (Sweden)

    César Humberto Guzmán Valdivia

    2013-07-01

    Full Text Available Engineering education in mechatronics is an attractive field of research because it is a new multidisciplinary career. However, a potential problem is the reprobation rate. In the period from January to April 2012 at the Universidad Politécnica de Zacatecas a 53% regular students of a total of 197 were registered. To find the causes of this problem, a survey was conducted to determine the causes of reprobation, lack of motivation and interest to a population of 96 students, of which 40 were the first training cycle, 32 the second and 24 the third. The surveys yielded three main results. The first indicates that the lack of interest is proportional to the time spent in college. The second shows that the reprobation rate is linked to the laziness and the excess of courses. And the last shows a lack of motivation and low expectations of student due to the monotony of the theoretical courses. In conclusion, more research is needed to have a motivated student in an engineering career in mechatronics.

  10. The subjetivacion of the lack: between Lacan and Hegel

    Directory of Open Access Journals (Sweden)

    Lorena Souyris Oportot

    2014-05-01

    Full Text Available The present article develops a reflection concerning the figure of the subjectivation and the statute of the lack  in relation to Jacques Lacan y Hegel's thought . The analysis will be addressed from a philosophical approach as and with a psychoanalytic perspective, to show the need to understand the subjectivity, not already as a "work" of duel, but ligature to the loss and the split. The idea is that the above mentioned significances make possible deconstruir and to rethink the duel in lack, that he structures to the subject in an experience "escripturaire" (escriptural and, for the same thing, of dispossession. So that the figure of the subjetivación "in" lack  will allow to grant an important place to the non-place while I spread where the unthinkable thing and the "Autre" registers.  Once exposed this, the reflection will focus on the tragic exigences behind experience “escripturaire” expressed in the image of Antigone

  11. Golgi disruption and early embryonic lethality in mice lacking USO1.

    Directory of Open Access Journals (Sweden)

    Susie Kim

    Full Text Available Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development.

  12. Meiosis I in Xenopus oocytes is not error-prone despite lacking spindle assembly checkpoint.

    Science.gov (United States)

    Liu, Dandan; Shao, Hua; Wang, Hongmei; Liu, X Johné

    2014-01-01

    The spindle assembly checkpoint, SAC, is a surveillance mechanism to control the onset of anaphase during cell division. SAC prevents anaphase initiation until all chromosome pairs have achieved bipolar attachment and aligned at the metaphase plate of the spindle. In doing so, SAC is thought to be the key mechanism to prevent chromosome nondisjunction in mitosis and meiosis. We have recently demonstrated that Xenopus oocyte meiosis lacks SAC control. This prompted the question of whether Xenopus oocyte meiosis is particularly error-prone. In this study, we have karyotyped a total of 313 Xenopus eggs following in vitro oocyte maturation. We found no hyperploid egg, out of 204 metaphase II eggs with countable chromosome spreads. Therefore, chromosome nondisjunction is very rare during Xenopus oocyte meiosis I, despite the lack of SAC.

  13. Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus;

    2002-01-01

    A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity....../Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte...... differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role...

  14. Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant

    Science.gov (United States)

    Kazama, Makoto; Ogiwara, Sanae; Makiuchi, Takashi; Yoshida, Kazuhiro; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi; Tachibana, Hiroshi

    2017-01-01

    The anaerobic protozoan parasite Entamoeba histolytica has mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in mitosomes is not uniform, and how mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for mitosomes with hemagglutinin-tagged protein into recipient cells containing mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor mitosomes may obtain recipient proteins through fusion with other mitosomes or through de novo synthesis of proteins in recipient cells. PMID:28287148

  15. Leishmania promastigotes lack phosphatidylserine but bind annexin V upon permeabilization or miltefosine treatment.

    Directory of Open Access Journals (Sweden)

    Adrien Weingärtner

    Full Text Available The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31P nuclear magnetic resonance (NMR spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

  16. LACK OF NOTIFICATION OF COMPULSORY NOTIFICATION DISEASES IN HOSPITAL SETTINGS

    Directory of Open Access Journals (Sweden)

    Rubens Griep

    2004-04-01

    Full Text Available This research encompasses the diseases of compulsory lack of notification inhospital settings and its interface with the Health Information System (Sistema de Informaçãode Saúde – SIS developed and implemented by the Welfare System in Brazil (Sistema Únicode Saúde – SUS. It tries to identify the elements epidemiology is based on, referring to itsaspects as basis for the development of epidemiologic monitoring actions of contagiousdiseases in the country. It focuses on the following question: what are the factors thatcontribute and/or determine the flaws in the process of notification for compulsory notificationdiseases? The results obtained through a questionnaire presented to the personnel responsiblefor the Hospital Infection Control Service (Serviço de Controle de Infecção Hospitalar showtheir lack of preparedness and technical knowledge, as well as their team’s, in relation to thedynamic functioning of the Epidemiologic Vigilance Service (Serviço de VigilânciaEpidemiológica. It points to the flaws due to the influence exerted by the public and/or privatecharacter of the institutions and considers the possibility of lack of commitment and responsibility of the multi-professional team in the maintenance of the preestablished flow. As aproposal, we present an adoption of continuous educational actions through the implementationof a Long Distance Post Graduation course, aiming for the development of new possibilities forthe teaching-learning process, characterized by the ongoing quest for new knowledge and focuson the student. The implementation of a local and municipal Permanent Habilitation Programmay complement the need for updating, as well as make the discussion of the cases and dataof the reality possible, thus aiming to adopt joined measures in order to cope with the presentedepidemiologic situations.

  17. Lack of Syneresis during Gelation of Dense Colloidal Suspensions.

    Science.gov (United States)

    Helbig; Hütter; Schönholzer

    2000-02-01

    This study reports experimental results about the shrinkage of particle networks produced by pH-induced destabilization of dense colloidal suspensions. The resulting solid networks exhibit no syneresis effects, at least prior to aging of the gel. From this lack of syneresis it is concluded that the solidification in wet particle systems either is not purely determined by energy (but is also influenced by entropic effects) or cannot be explained within the framework of (static) equilibrium thermodynamics at all. Copyright 2000 Academic Press.

  18. Increased sensitivity to kindling in mice lacking TSP1.

    Science.gov (United States)

    Mendus, D; Rankin-Gee, E K; Mustapha, M; Porter, B E

    2015-10-01

    The development of a hyperexcitable neuronal network is thought to be a critical event in epilepsy. Thrombospondins (TSPs) regulate synaptogenesis by binding the neuronal α2δ subunit of the voltage-gated calcium channel. TSPs regulate synapse formation during development and in the mature brain following injury. It is unclear if TSPs are involved in hyperexcitability that contributes to the development of epilepsy. Here we explore the development of epilepsy using a pentylenetetrazole (PTZ) kindling model in mice lacking TSP1 and TSP2. Unexpectedly, we found increased sensitivity to PTZ kindling in mice lacking TSP1, while mice lacking TSP2 kindled similar to wild-type. We found that the increased seizure susceptibility in the TSP1 knockout (KO) mice was not due to a compensatory increase in TSP2 mRNA as TSP1/2 KO mice were sensitive to PTZ, similar to the TSP1 KO mice. Furthermore, there were similar levels of TGF-B signal activation during kindling in the TSP1 KO mice compared to wild-type. We observed decreased expression of voltage-dependent calcium channel subunit CACNA2D1 mRNA in TSP1, TSP2, and TSP1/2 KO mice. Decreased CACNA2D2 mRNA was only detected in mice that lacked TSP1 and α2δ-1/2 protein levels in the cortex were lower in the TSP 1/2 KO mice. CACNA2D2 knockout mice have spontaneous seizures and increased PTZ seizure susceptibility. Here we report similar findings, TSP1, and TSP1/2 KO mice have low levels of CACNA2D2 mRNA expression and α2δ-1/2 receptor level in the cortex, and are more susceptible to seizures. CACNA2D2 mutations in mice and humans can cause epilepsy. Our data suggest TSP1 in particular may control CACNA2D2 levels and could be a modifier of seizure susceptibility.

  19. The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis

    Directory of Open Access Journals (Sweden)

    Barth Julia MI

    2012-12-01

    Full Text Available Abstract Background The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC, but not in the germline cells (GCs. However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development. Results Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling. Conclusion Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline and signal receiving cell (FC, thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

  20. Possible mechanisms of lack of dentin bridge formation in response to calcium hydroxide in primary teeth

    Directory of Open Access Journals (Sweden)

    G R Ravi

    2015-01-01

    Full Text Available Introduction: The usage of Calcium hydroxide (CaOH2 has wide applications due to the property of osteo-inductive, protective, and antibacterial actions. However, it is not used in primary teeth, as it fails to form reparative dentin and the exact mechanism has not been explained. The hypothesis: The authors propose an explanation that lack of dentin bridge formation in response to (CaOH2 in primary teeth could be multifactorial: inability of the deciduous stem cells to generate complete dentin-pulp-like tissue; the absence of calcium-magnesium-dependent adenosine triphosphatase (Ca-Mg ATPase in the odontoblasts; the pre-existing predilection of deciduous dentine pulp to form odontoclasts; the solubility of (CaOH2. Evaluation of the hypothesis: The hypothesis discusses the innate traits of the deciduous stem cells that lack the ability to form the dentin bridge, the absence of Ca-Mg ATPase enzyme and increased solubility of (CaOH2 together fail to stimulate the odontoblasts. Alternatively, pre-existing progenitor cells with proclivity to change into odontoclasts may cause internal resorption and hamper formation of reparative dentin.

  1. Nonadherence is Associated with Lack of HIV-Related Knowledge

    DEFF Research Database (Denmark)

    Dyrehave, Charlotte; Rasmussen, Dlama Nggida; Hønge, Bo Langhoff

    2016-01-01

    -sectional study included 494 HIV-infected individuals from the Bissau HIV Cohort in Guinea-Bissau. They completed a questionnaire designed for assessment of adherence and HIV-related knowledge. RESULTS: A majority were female, 41% were illiterate, 25% did not take the medicine during the last 4 days, and 23......BACKGROUND: Poor treatment adherence is a main barrier for effective antiretroviral therapy (ART) globally. HIV-related knowledge may affect understanding and utilization of HIV medical information, hence limited health literacy is a known barrier to treatment adherence. DESIGN AND METHODS: A cross......% skipped their medicine during weekends. The most frequent reasons for not taking medicine were simply forgetting, side effects, lack of food, and being too ill to attend the clinic. Nonadherent patients had a lower level of HIV-related knowledge. CONCLUSION: Main barriers for nonadherence were side...

  2. Lack of time management as a psychosocial work risk

    Directory of Open Access Journals (Sweden)

    Ramon Cladellas

    2008-10-01

    Full Text Available This paper is aimed to explore the possible relationship between workers' lack of time management and several psychosocial risks. The psychosocial risks were assessed by means of the ISTAS21 Questionnaire, the Spanish version of the CoPsoQ (Copenhagen Psychological Questionnaire. More specifically, nine dimensions, which are directly related with time management, satisfaction, health and stress, were selected for evaluation. Time management was measured through the following variables: quantitative demands, influences and control of the time. Drawing on a sample of 142 workers from four departments (development, implantation, support and administration, the research results show that the employees who belong to a department that offers few opportunities for individual time management are less satisfied, have worse general and mental health, and experience more behavioral, symptomatic and cognitive stress than those who can manage their work schedule.

  3. The lack of carbon stars in the Galactic bulge

    Institute of Scientific and Technical Information of China (English)

    Zhu Chun-Hua; Lv Guo-Liang; Wang Zhao-Jun; Zhang Jun

    2008-01-01

    In order to explain the lack of carbon stars in the Galactic bulge, we have made a detailed study of thermal pulseasymptotic giant branch (TP-AGB) stars by using a population synthesis code. The effects of the oxygen overabundance and the mass loss rate on the ratio of the number of carbon stars to that of oxygen stars in the Galactic bulge are discussed. We find that the oxygen overabundance which is about twice as large as that in the solar neighbourhood (close to the present observations) is insufficient to explain the rareness of carbon stars in the bulge. We suggest that the large mass loss rate may serve as a controlling factor in the ratio of the number of carbon stars to that of oxygen stars.

  4. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart

    Directory of Open Access Journals (Sweden)

    Wenjing Xu

    2015-10-01

    Full Text Available Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1 might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  5. When innovative instructional designs are too innovative: lack of schema

    DEFF Research Database (Denmark)

    Kjærgaard, Thomas; Wahl, Christian

    2015-01-01

    and it was developed to motivate and encourage the students to engage in more situated learning processes. The course is infamous for low attendance and for demotivating the students; hence the new instructional design should motivate students to attend the lessons and to participate. The new instructional design...... utilized teacher-produced video clips to qualify the students learning in the preparation for the lessons and new pedagogical activities during lessons to make the learning process more situated. However, the outcome was not as planned. The students didn’t recognize the video clips as a significant part...... of the preparation for the lessons and therefore they were not prepared for the activities that the teacher had planned for the lessons. The paper investigates the students’ lack of ‘schema’ and suggests ways of developing schemata to learn in a situated learning environment....

  6. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart.

    Science.gov (United States)

    Xu, Wenjing; Barrientos, Tomasa; Mao, Lan; Rockman, Howard A; Sauve, Anthony A; Andrews, Nancy C

    2015-10-20

    Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  7. Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction.

    Science.gov (United States)

    Li, Nan; Liu, Zhenghui; Zhang, Yue; Chen, Qiaoyuan; Liu, Peng; Cheng, C Yan; Lee, Will M; Chen, Yongmei; Han, Daishu

    2015-03-01

    The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double-knockout (Axl(-/-)Mer(-/-)) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freund's adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testes, impaired permeability of the blood-testis barrier and generation of autoantibodies against germ cell antigens in Axl(-/-)Mer(-/-) mice. Immunization also induced mild EAO in Axl or Mer single-gene-knockout mice. By contrast, a single immunization failed to induce EAO in wild-type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.

  8. Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

    Directory of Open Access Journals (Sweden)

    Min Yu

    Full Text Available BACKGROUND: Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined. METHODOLOGY/PRINCIPAL FINDINGS: Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia. CONCLUSIONS: Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

  9. Generation of mice lacking DUF1220 protein domains

    DEFF Research Database (Denmark)

    Keeney, J G; O'Bleness, M S; Anderson, N

    2015-01-01

    , fasting glucose levels, reduce sodium levels in male mutants, increased levels of the liver functional indicator ALAT/GPT in males, levels of alkaline phosphatase (also an indicator of liver function), mean R and SR amplitude by electrocardiography, elevated IgG3 levels, a reduced ratio of CD4:CD8 cells...

  10. Epidermal hyperplasia and appendage abnormalities in mice lacking CD109.

    Science.gov (United States)

    Mii, Shinji; Murakumo, Yoshiki; Asai, Naoya; Jijiwa, Mayumi; Hagiwara, Sumitaka; Kato, Takuya; Asai, Masato; Enomoto, Atsushi; Ushida, Kaori; Sobue, Sayaka; Ichihara, Masatoshi; Takahashi, Masahide

    2012-10-01

    CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

  11. Clinical errors as a lack of context responsiveness.

    Science.gov (United States)

    Bugatti, Matteo; Boswell, James F

    2016-09-01

    Although standardized treatments have the potential to decrease clinical errors, within-session responsiveness is complicated and complementary frameworks may be needed to foster enhanced responsiveness in the context of evidence-based treatments. Recent efforts have targeted the enhancement of flexibility and responsiveness in the delivery of manualized treatments, including the development of transdiagnostic treatments (i.e., protocols that are designed to be used across different diagnoses) intended to tailor intervention principles to the needs of individual patients. Context-Responsive Psychotherapy Integration (Constantino, Boswell, Bernecker, & Castonguay, 2013) offers an framework that supports the utilization of evidence-based clinical strategies in response to the identification of specific process markers. Failure to identify or appropriately respond to such markers may result in negative therapeutic process as well as outcomes. This case study uses the context-response psychotherapy integration framework to understand critical moments of clinical decision-making through examining an individual treatment case that unilaterally terminated after seven sessions of transdiagnostic treatment. This illustrative empirical case analysis focuses on three potential clinical errors, as indicated by a lack of responsiveness to three candidate process markers: (a) low outcome expectations, (b) self-strivings, and (c) outcome monitoring. For each clinical error, alternative clinical strategies are discussed. (PsycINFO Database Record

  12. Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

    Science.gov (United States)

    Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S

    2009-12-01

    The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

  13. Lack of a Benign Interpretation Bias in Social Anxiety Disorder

    Science.gov (United States)

    Amir, Nader; Prouvost, Caroline; Kuckertz, Jennie M.

    2013-01-01

    Cognitive models of social anxiety posit that recurrent interpretation of ambiguous information as threatening maintains symptoms (e.g. Clark & Wells, 1995, pp. 69–93, Social phobia: Diagnosis, assessment, and treatment. New York: Guilford Press; Rapee & Heimberg, 1997, pp. 741–756, Behavior Research and Therapy, 35). However, biased interpretation may also be represented as a failure to make a benign interpretation of the ambiguous event. Furthermore, interpretation bias can be characterized by both an online (automatic) component and an offline (effortful) component (Hirsch & Clark, 2004, pp. 799–825, Clinical Psychology Review, 24). To measure both benign and threat biases, as well as examine the effect of social anxiety on offline versus online interpretations, Beard and Amir (2009, pp. 1135–1141, Behaviour Research and Therapy, 46) developed the Word Sentence Association Paradigm (WSAP). In the current study, we administered the WSAP to a group of participants diagnosed with social anxiety disorder (SAD) as well as to a group of non-anxious control (NAC) participants. We found that participants with SAD demonstrated a lack of benign online bias, but not an online threat bias when compared to NACs. However, when examining offline biases, SAD patients endorsed social threat interpretations and rejected benign social interpretations to a greater degree than non-anxious individuals. Our results, when taken together, clearly implicate the role of reduced bias toward benign information in SAD. PMID:22545788

  14. Vulnerability to schizophrenia and lack of common sense.

    Science.gov (United States)

    Stanghellini, G

    2000-01-01

    This article explores the hypothesis that the relational deficit in schizophrenia is not a consequence of acute symptoms and course but instead is a fundamental aspect of schizophrenic vulnerability. This basic relational deficit could be better understood as disconnectedness from common sense. Common sense is a tool for adaptation whose main scope is establishing cause-and-effect and motivational relationships in the physical and social realms. The common sense deficit appears to involve a lack of intuitive attunement (impaired capacity to accurately typify the mental states of other persons because of the incapacity to be involved in their mental lives) and a damaged social knowledge network (disorders of the background of knowledge useful for organizing everyday experiences). Three dimensions of schizophrenic vulnerability can be distinguished: the sensory, conceptualization, and attitudinal dimensions. Sensory disorders are aberrations of self, body, and world perceptions. Conceptualization disorders are disturbances in the attribution of meanings and intentions. Attitudinal disorders consist of eccentricities in the individual's structure of values and beliefs, characterized by distrust toward conventional knowledge and attunement. This article describes the present state and possible future directions of qualitative analyses and empirical investigations relevant to assessing the interplay between vulnerability dimensions and disorders of common sense.

  15. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  16. New graduate nurses' experiences about lack of professional confidence.

    Science.gov (United States)

    Ortiz, Jennifer

    2016-07-01

    Professional confidence is an essential trait for new graduate nurses to possess in order to provide quality patient care in today's complex hospital setting. However, many new graduates are entering the workforce without it and this remains to be explored. This study describes how new graduate nurses accounted for their lack of professional confidence upon entry into professional practice and how it developed during their first year of practice in the hospital setting. Two face-to-face, individual interviews of 12 participants were utilized to capture the lived experiences of new graduate nurses to gain an understanding of this phenomenon. After manual content analysis seven themes emerged: communication is huge, making mistakes, disconnect between school and practice, independence, relationship building, positive feedback is important, and gaining experience. The findings indicate that the development of professional confidence is a dynamic process that occurs throughout the first year of practice. New graduate nurses must experience both positive and negative circumstances in order to move toward the attainment of professional confidence. Knowing this, nurse educators in academia as well as in the hospital setting may better support the development of professional confidence both before and during the first year of practice.

  17. Lack of fiscal discipline: Challenges and possible solutions

    Directory of Open Access Journals (Sweden)

    Ćirović Nikola

    2013-01-01

    Full Text Available The lack of fiscal discipline is one of the most pressing issues in the framework of various economic systems today. The consequences of the global economic crisis have shown all the weaknesses of measures and mechanisms in terms of decision-making in fiscal policy. This problem can be seen now in almost every country. The problem of inadequate implementation of fiscal policy and the maintenance of fiscal discipline is reflected in the fact that their consequences are not visible right away, but tend to show their negative effects much later. Today, we have a situation where a certain current government suffers badly implemented prior fiscal discipline of a previous government. The paper identified causes of fiscal indiscipline, which is most easily seen in the budget process. In addition to determining the causes of fiscal indiscipline, the author puts forward a set of different possible solutions that could positively affect the stabilization of the growing public debt and eliminate the cause of fiscal indiscipline. Possible solutions are analyzed, as well as the positive and negative aspects of their use, and possible implementation problems of specific solutions.

  18. Lack of global epigenetic methylation defects in CBS deficient mice.

    Science.gov (United States)

    Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming; Gupta, Sapna; Slifker, Michael J; Li, Yue-Sheng; Andrews, Andrew J; Kruger, Warren D

    2017-01-01

    Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

  19. Virus Spread and Immune Response Following Anterior Chamber Inoculation of HSV-1 Lacking the Beclin-Binding Domain (BBD)

    OpenAIRE

    ZHANG, Ming; Covar, Jason; Zhang, Nancy Y.; Chen, Wen; Marshall, Brendan; Mo, Juan; Atherton, Sally S.

    2013-01-01

    The autophagy response induced by HSV-1 infection is antagonized by the Beclin-binding domain (BBD). The purpose of this study was to determine if lack of the BBD affects viral spread and immune response in the eyes and brain. Our results showed that lack of the BBD increases autophagy response and activation of NLRP3 imflammasome, which in turn induces a more rapid innate immune response mediated by macrophage/microglia and NK cells in the injected eye, limiting virus replication and retinal...

  20. Commercially available antibodies against human and murine histamine H₄-receptor lack specificity.

    Science.gov (United States)

    Beermann, Silke; Seifert, Roland; Neumann, Detlef

    2012-02-01

    Antibodies are important tools to detect expression and localization of proteins within the living cell. However, for a series of commercially available antibodies which are supposed to recognize G-protein-coupled receptors (GPCR), lack of specificity has been described. In recent publications, antisera against the histamine H₄-receptor (H₄R), which is a member of the GPCR family, have been used to demonstrate receptor expression. However, a comprehensive characterization of these antisera has not been performed yet. Therefore, the purpose of our study was to evaluate the specificity of three commercially available H₄R antibodies. Sf9 insect cells and HEK293 cells expressing recombinant murine and human H₄R, spleen cells obtained from H₄⁻/⁻ and from wild-type mice, and human CD20⁺ and CD20⁻ peripheral blood cells were analyzed by flow cytometry and Western blot using three commercially available H₄R antibodies. Our results show that all tested H₄R antibodies bind to virtually all cells, independently of the expression of H₄R, thus in an unspecific fashion. Also in Western blot, the H₄R antibodies do not bind to the specified protein. Our data underscore the importance of stringent evaluation of antibodies using valid controls, such as cells of H₄R⁻/⁻ mice, to show true receptor expression and antigen specificity. Improved validation of commercially available antibodies prior to release to the market would avoid time-consuming and expensive validation assays by the user.

  1. Cerebellar deficits and hyperactivity in mice lacking Smad4.

    Science.gov (United States)

    Zhou, Yong-Xing; Zhao, Mingrui; Li, Dan; Shimazu, Kazuhiro; Sakata, Kazuko; Deng, Chu-Xia; Lu, Bai

    2003-10-24

    Smad4 is a central mediator of TGF-beta signals, which are known to play essential roles in many biological processes. Using a Cre-loxP approach to overcome early embryonic lethality, we have studied functions of TGF-beta/Smad4 signals in the central nervous system (CNS). No obvious deficits were detected in mice carrying the targeted disruption of Smad4 in the CNS. The overall morphology of the hippocampus appeared normal. There was no change in the proliferation of neuronal precursor cells, nor in several forms of synaptic plasticity. In contrast, deletion of Smad4 resulted in a marked decrease in the number of cerebellar Purkinje cells and parvalbumin-positive interneurons. Accompanied by the abnormality in the cerebellum, mutant mice also exhibited significantly increased vertical activity. Thus, our study reveals an unexpected role for Smad4 in cerebellar development and in the control of motor function.

  2. Lack of response to unaligned chromosomes in mammalian female gametes.

    Science.gov (United States)

    Sebestova, Jaroslava; Danylevska, Anna; Novakova, Lucia; Kubelka, Michal; Anger, Martin

    2012-08-15

    Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy

  3. Lack of Cholesterol Awareness among Physicians Who Smoke

    Directory of Open Access Journals (Sweden)

    Richard E. Scranton

    2009-02-01

    Full Text Available Cigarette use is a known risk factor for the development of coronary artery disease (CAD as it adversely affects HDL cholesterol levels and promotes thrombogenesis. Smoking may also be associated with behavioral characteristics that potentiate the risk of CAD. A lack of cholesterol knowledge would indicate an aversion to a prevention-oriented lifestyle. Thus, our goal was to determine the association between tobacco use and knowledge of self-reported cholesterol among male physicians. Using the 1982 and follow-up questionnaires from the physician health study, we report the changes in the frequencies of awareness of self-reported total cholesterol and cardiovascular risk factors among the 22,067 participants. We classified physicians as being aware of their cholesterol if they reported a cholesterol level and unaware if the question was left unanswered. In 1997, 207 physicians were excluded, as the recorded cholesterol was not interpretable, leaving 21,860 for our follow up analyses. Using unadjusted logistic models, we determined the odds ratios (OR and 95% confidence intervals (CI of not reporting a cholesterol level in either 1982 or 1997 for each specified risk factor. We then evaluated whether the lack of cholesterol awareness at both time points was associated with the use of tobacco throughout the study. After 14-years of follow up, cholesterol awareness increased from 35.9 to 58.6 percent. During this period, the frequency of hypertension and hyperlipidemia treatment increased (13.5 to 40.5% and 0.57% to 19.6% respectively, as did the diagnosis of diabetes (2.40 to 7.79%. Behavioral characteristics such as a sedentary lifestyle and obesity also increased (27.8 to 42% and 43.5 to 53.5%, respectively, however the proportion of current smokers deceased from 11.1 to 4.05%. The percentages of individuals being unaware of their cholesterol decreased in all risk factor groups. However, individuals were likely to be unaware of their cholesterol

  4. Lack of association between postactivation potentiation and subsequent jump performance.

    Science.gov (United States)

    Pearson, Stephen John; Hussain, Syed Robiul

    2014-01-01

    Postactivation potentiation (PAP) is a strategy that has been used to acutely enhance the performance of explosive activities. Although, isometric maximal voluntary contractions (MVCs) have previously been shown to enhance subsequent explosive performance, no information currently exists regarding (1) the optimal variables (intensity/volume) of a MVC that best elicits a PAP response, and (2) the utilisation of evoked isometric twitch contractions in combination with performance measures to directly ascertain the presence of PAP following a MVC, and its relationship to performance. Thus, the purpose of this study was to (1) investigate the influence of isometric contraction duration on the PAP response, and (2) to determine the relationship between PAP, indicated as potentiation of muscle twitch force and subsequent jump performance following different-duration MVCs. Eight males (age: 21 ± 0.99) were assessed using performance measures [countermovement jumps] and evoked twitch contractions, before and 4 minutes after three different conditioning contractions (CCs), (1) a 3-second MVC (MVC3), (2) a 5-second MVC (MVC5) and (3) a 7-second MVC (MVC7). Following all CCs, peak twitch torque of the knee extensor muscles was found to increase (MVC3, + 3.9%; MVC5, + 9.6%; MVC7, + 5.2%), although not significantly (P > 0.05). No significant increases in jump height, jump power, rate of force development or takeoff velocity were observed following any of the CCs (P > 0.05). There was also a lack of association between the changes in PAP (twitch torque) and jump height following all CCs (MVC3, r = 0.25; MVC5, r = 0.28; MVC7, r = -0.47). These data indicate that PAP as assessed via twitch contractions is not associated with performance measures subsequent to single-set isometric CCs of varying durations.

  5. Implications of the lack of desiccation tolerance in recalcitrant seeds

    Directory of Open Access Journals (Sweden)

    Patricia eBerjak

    2013-11-01

    Full Text Available A suite of interacting processes and mechanisms enables tolerance of desiccation and storage (conservation of orthodox seeds in the dry state. While this is a long-term option under optimised conditions, dry orthodox seeds are not immortal, with life spans having been characterised as short, intermediate and long. Factors facilitating desiccation tolerance are metabolic ‘switch-off’ and intracellular dedifferentiation. Recalcitrant seeds lack these mechanisms, contributing significantly to their desiccation sensitivity.Consequently, recalcitrant seeds, which are shed at high water contents, can be stored only in the short-term, under conditions not allowing dehydration. The periods of such hydrated storage are constrained by germination that occurs without the need for extraneous water, and the proliferation of seed-associated fungi. Cryopreservation is viewed as the only option for long-term conservation of the germplasm of recalcitrant-seeded species. This is not easily achieved, as each of the necessary procedures imposes oxidative damage. Intact recalcitrant seeds cannot be cryopreserved, the common practice being to use excised embryos or embryonic axes as explants. Dehydration is a necessary procedure prior to exposure to cryogenic temperatures, but this is associated with metabolism-linked injury mediated by uncontrolled ROS generation and failing anti-oxidant systems. While the extent to which this occurs can be curtailed by maximising drying rate (flash drying it cannot be completely obviated. Explant cooling for, and rewarming after, cryostorage must necessarily be rapid, to avoid ice crystallisation. The ramifications of desiccation sensitivity are discussed, as are problems involved in cryostorage, particularly those accompanying dehydration and damage consequent upon ice crystallisation. While desiccation sensitivity is a ‘fact’ of seed recalcitrance, resolutions of the difficulties involved germplasm conservation are

  6. Lack of plasma kallikrein-kinin system cascade in teleosts.

    Directory of Open Access Journals (Sweden)

    Marty Kwok-Shing Wong

    Full Text Available The kallikrein-kinin system (KKS consists of two major cascades in mammals: "plasma KKS" consisting of high molecular-weight (HMW kininogen (KNG, plasma kallikrein (KLKB1, and bradykinin (BK; and "tissue KKS" consisting of low molecular-weight (LMW KNG, tissue kallikreins (KLKs, and [Lys(0]-BK. Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg(0]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade.

  7. Lack of glucagon receptor signaling and its implications beyond glucose homeostasis.

    Science.gov (United States)

    Charron, Maureen J; Vuguin, Patricia M

    2015-03-01

    Glucagon action is transduced by a G protein-coupled receptor located in liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart, pancreatic β-cells, and placenta. Genetically modified animal models have provided important clues about the role of glucagon and its receptor (Gcgr) beyond glucose control. The PubMed database was searched for articles published between 1995 and 2014 using the key terms glucagon, glucagon receptor, signaling, and animal models. Lack of Gcgr signaling has been associated with: i) hypoglycemic pregnancies, altered placentation, poor fetal growth, and increased fetal-neonatal death; ii) pancreatic glucagon cell hyperplasia and hyperglucagonemia; iii) altered body composition, energy state, and protection from diet-induced obesity; iv) impaired hepatocyte survival; v) altered glucose, lipid, and hormonal milieu; vi) altered metabolic response to prolonged fasting and exercise; vii) reduced gastric emptying and increased intestinal length; viii) altered retinal function; and ix) prevention of the development of diabetes in insulin-deficient mice. Similar phenotypic findings were observed in the hepatocyte-specific deletion of Gcgr. Glucagon action has been involved in the modulation of sweet taste responsiveness, inotropic and chronotropic effects in the heart, satiety, glomerular filtration rate, secretion of insulin, cortisol, ghrelin, GH, glucagon, and somatostatin, and hypothalamic signaling to suppress hepatic glucose production. Glucagon (α) cells under certain conditions can transdifferentiate into insulin (β) cells. These findings suggest that glucagon signaling plays an important role in multiple organs. Thus, treatment options designed to block Gcgr activation in diabetics may have implications beyond glucose homeostasis.

  8. The Drosophila Anion Exchanger (DAE lacks a detectable interaction with the spectrin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Base Christine

    2010-06-01

    Full Text Available Abstract Background Current models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1 was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE as a marker for studies of the downstream effects of spectrin cytoskeleton mutations. Results Sequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila. Conclusions Based on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

  9. Increased renal renin content in mice lacking the Na+/H+ exchanger NHE2.

    Science.gov (United States)

    Hanner, Fiona; Chambrey, Régine; Bourgeois, Soline; Meer, Elliott; Mucsi, István; Rosivall, László; Shull, Gary E; Lorenz, John N; Eladari, Dominique; Peti-Peterdi, János

    2008-04-01

    Macula densa (MD) cells express the Na(+)/H(+) exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2(+/+)) and NHE2 knockout (NHE2(-/-)) mice. Immunofluorescence studies indicated that NHE2(-/-) mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2(-/-) mice was increased approximately 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2(-/-) mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2(+/+) mice, but the response was blunted in NHE2(-/-) mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2(-/-) mice compared with wild type. NHE2(-/-) mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2(-/-) kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE(2)-renin release pathway.

  10. Lack of malate valve capacities lead to improved N-assimilation and growth in transgenic A. thaliana plants.

    Science.gov (United States)

    Selinski, Jennifer; Scheibe, Renate

    2014-01-01

    In this study we analyzed the relationship between malate valve capacities, N-assimilation, and energy metabolism. We used transgenic plants either lacking the chloroplast NADP-dependent malate dehydrogenase or mutants with a decreased transcript level of the plastid-localized NAD-dependent malate dehydrogenase. Plants were grown on nitrate or ammonium, respectively, as the sole N-source and transcripts were analyzed by qRT-PCR. We could show that the lack of malate valve capacities enhances N-assimilation and plastidial glycolysis by increasing transcript levels of Fd-GOGATs or NADH-GOGAT and plastidic NAD-GAPDHs (GapCps), respectively. Based on our results, we conclude that the lack of malate valve capacities is balanced by an increase of the activity of plastid-localized glycolysis in order to cover the high demand for plastidial ATP, stressing the importance of the plastids for energy metabolism in plant cells.

  11. Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity

    Directory of Open Access Journals (Sweden)

    Schwartz Robert J

    2007-11-01

    Full Text Available Abstract Background Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs, it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects 123. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis. Results We have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage, heart tube looping (28-somite stage, and outflow track septation (38-somite stage. Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation. Conclusion The results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and

  12. Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle.

    Science.gov (United States)

    Barrientos, Tomasa; Laothamatas, Indira; Koves, Timothy R; Soderblom, Erik J; Bryan, Miles; Moseley, M Arthur; Muoio, Deborah M; Andrews, Nancy C

    2015-11-01

    Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid β oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders.

  13. Absence of venous valves in mice lacking Connexin37.

    Science.gov (United States)

    Munger, Stephanie J; Kanady, John D; Simon, Alexander M

    2013-01-15

    Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.

  14. Lack of functional specialization of neurons in the mouse primary visual cortex that have expressed calretinin

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    Daniela eCamillo

    2014-09-01

    Full Text Available Calretinin is a calcium-binding protein often used as a marker for a subset of inhibitory interneurons in the mammalian neocortex. We studied the labeled cells in offspring from a cross of a Cre-dependent reporter line with the CR-ires-Cre mice, which express Cre-recombinase in the same pattern as calretinin. We found that in the mature visual cortex, only a minority of the cells that have expressed calretinin and Cre-recombinase during their lifetime is GABAergic and only about 20% are immunoreactive for calretinin. The reason behind this is that calretinin is transiently expressed in many cortical pyramidal neurons during development. To determine whether neurons that express or have expressed calretinin share any distinct functional characteristics, we recorded their visual response properties using GCaMP6s calcium imaging. The average orientation selectivity, size tuning, and temporal and spatial frequency tuning of this group of cells, however, match the response profile of the general neuronal population, revealing the lack of functional specialization for the features studied.

  15. Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, Jennifer L. [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Diamond, Michael S., E-mail: diamond@borcim.wustl.edu [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110 (United States); Pathology & Immunology, Washington University School of Medicine, St Louis., MO 63110 (United States); The Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis., MO 63110 (United States)

    2015-05-15

    N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on their RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.

  16. A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine

    DEFF Research Database (Denmark)

    Bergström, N; Jansson, P.-E.; Kilian, Mogens

    2003-01-01

    to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double......Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast...... choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four....

  17. A stop codon mutation in SCN9A causes lack of pain sensation.

    Science.gov (United States)

    Ahmad, Sultan; Dahllund, Leif; Eriksson, Anders B; Hellgren, Dennis; Karlsson, Urban; Lund, Per-Eric; Meijer, Inge A; Meury, Luc; Mills, Tracy; Moody, Adrian; Morinville, Anne; Morten, John; O'donnell, Dajan; Raynoschek, Carina; Salter, Hugh; Rouleau, Guy A; Krupp, Johannes J

    2007-09-01

    The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.

  18. Lack of association of TIM3 polymorphisms and allergic phenotypes

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    Laprise Catherine

    2009-06-01

    Full Text Available Abstract Background T-cell immunoglobulin mucin-3 (TIM3 is a TH1-specific type 1 membrane protein that regulates TH1 proliferation and the development of immunological tolerance. TIM3 and its genetic variants have been suggested to play a role in regulating allergic diseases. Polymorphisms in the TIM3 promoter region have been reported to be associated with allergic phenotypes in several populations. The aims of this study were to examine whether genetic variation in the promoter region of TIM3 influenced transcription of the gene and risk for allergic phenotypes. Methods We performed 5' rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. We screened for polymorphisms in the promoter region. Deletion analysis was used to localize the promoter region of TIM3. Genotyping was performed by TaqMan assays in three asthma/allergy population samples. Results We found two regions with promoter activity in TIM3. One region was from -214 bp to +58 bp and the other from -1.6 kb to -914 bp relative to the transcription start site. None of the single nucleotide polymorphisms (SNPs or haplotypes affected the transcriptional activity in reporter gene assays. No association between the SNPs and any phenotype was observed in the study cohorts. Conclusion Our findings indicate that SNPs and haplotypes in the TIM3 promoter region do not have a functional effect in vitro and are not associated with allergic diseases. These data suggest that polymorphisms in the TIM3 promoter region are unlikely to play an important role in susceptibility to allergic diseases.

  19. Lack of indication of myocardial cell damage after myocardial ischaemia in patients with severe stable angina

    DEFF Research Database (Denmark)

    Hansen, Knud Nørregaard; Egstrup, K; Nielsen, J R

    1992-01-01

    stenosis of one or more of the main coronary arteries and more than five ischaemic attacks per week. ST-segment monitoring was performed for 36 h. During the last 24 h of that period (period A) serial blood samples were analysed for myoglobin, CK and CK-MB using sensitive assays. Three days later (period B...

  20. Lack of promoting activity of four pesticides on induction of preneoplastic liver cell foci in rats.

    Science.gov (United States)

    Kato, T; Ogiso, T; Kato, K; Sano, M; Hasegawa, R; Shirai, T; Ito, N

    1995-01-01

    Four pesticides were examined for hepatopromoting activity using a medium-term bioassay based upon induction of glutathione S-transferase placental form (GST-P) positive foci in the rat liver. Male F344 rats were initially injected with diethylnitrosamine (DEN; 200 mg/kg body weight) intraperitoneally and 2 weeks later were treated with O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN; 75 and 150 ppm), diazinon (500 and 1,000 ppm), phenthoate (500 and 1,000 ppm), or iprobenfos (500 and 1,000 ppm) in the diet for 6 weeks and then killed, all rats being subjected to partial hepatectomy at week 3. All of the pesticides gave negative results, the numbers and areas of GST-P positive foci not exceeding the control values for animals given DEN alone. Indeed, a significant reduction of foci development was seen for EPN (75 ppm). These findings provide experimental evidence that the presently examined four pesticides do not have hepatocarcinogenic potential in rats.

  1. Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4.

    Science.gov (United States)

    Boettger, Thomas; Hübner, Christian A; Maier, Hannes; Rust, Marco B; Beck, Franz X; Jentsch, Thomas J

    2002-04-25

    Hearing depends on a high K(+) concentration bathing the apical membranes of sensory hair cells. K(+) that has entered hair cells through apical mechanosensitive channels is transported to the stria vascularis for re-secretion into the scala media(). K(+) probably exits outer hair cells by KCNQ4 K(+) channels(), and is then transported by means of a gap junction system connecting supporting Deiters' cells and fibrocytes() back to the stria vascularis. We show here that mice lacking the K(+)/Cl(-) (K-Cl) co-transporter Kcc4 (coded for by Slc12a7) are deaf because their hair cells degenerate rapidly after the beginning of hearing. In the mature organ of Corti, Kcc4 is restricted to supporting cells of outer and inner hair cells. Our data suggest that Kcc4 is important for K(+) recycling() by siphoning K(+) ions after their exit from outer hair cells into supporting Deiters' cells, where K(+) enters the gap junction pathway. Similar to some human genetic syndromes(), deafness in Kcc4-deficient mice is associated with renal tubular acidosis. It probably results from an impairment of Cl(-) recycling across the basolateral membrane of acid-secreting alpha-intercalated cells of the distal nephron.

  2. Exacerbation of diabetic renal alterations in mice lacking vasohibin-1.

    Directory of Open Access Journals (Sweden)

    Norikazu Hinamoto

    Full Text Available Vasohibin-1 (VASH1 is a unique endogenous inhibitor of angiogenesis that is induced in endothelial cells by pro-angiogenic factors. We previously reported renoprotective effect of adenoviral delivery of VASH1 in diabetic nephropathy model, and herein investigated the potential protective role of endogenous VASH1 by using VASH1-deficient mice. Streptozotocin-induced type 1 diabetic VASH1 heterozygous knockout mice (VASH1(+/- or wild-type diabetic mice were sacrificed 16 weeks after inducing diabetes. In the diabetic VASH1(+/- mice, albuminuria were significantly exacerbated compared with the diabetic wild-type littermates, in association with the dysregulated distribution of glomerular slit diaphragm related proteins, nephrin and ZO-1, glomerular basement membrane thickening and reduction of slit diaphragm density. Glomerular monocyte/macrophage infiltration and glomerular nuclear translocation of phosphorylated NF-κB p65 were significantly exacerbated in the diabetic VASH1(+/- mice compared with the diabetic wild-type littermates, accompanied by the augmentation of VEGF-A, M1 macrophage-derived MCP-1 and phosphorylation of IκBα, and the decrease of angiopoietin-1/2 ratio and M2 macrophage-derived Arginase-1. The glomerular CD31(+ endothelial area was also increased in the diabetic VASH1(+/- mice compared with the diabetic-wild type littermates. Furthermore, the renal and glomerular hypertrophy, glomerular accumulation of mesangial matrix and type IV collagen and activation of renal TGF-β1/Smad3 signaling, a key mediator of renal fibrosis, were exacerbated in the diabetic VASH1(+/- mice compared with the diabetic wild-type littermates. In conditionally immortalized mouse podocytes cultured under high glucose condition, transfection of VASH1 small interfering RNA (siRNA resulted in the reduction of nephrin, angiopoietin-1 and ZO-1, and the augmentation of VEGF-A compared with control siRNA. These results suggest that endogenous VASH1 may

  3. Lack of SIRPα phosphorylation and concomitantly reduced SHP-2-PI3K-Akt2 signaling decrease osteoblast differentiation.

    Science.gov (United States)

    Holm, Cecilia Koskinen; Engman, Sara; Sulniute, Rima; Matozaki, Takashi; Oldenborg, Per-Arne; Lundberg, Pernilla

    2016-09-09

    Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPα-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRPα-mutant cells. In conclusion, our data suggest that SIRPα signaling through SHP-2-PI3K-Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells.

  4. Impaired hair growth and wound healing in mice lacking thyroid hormone receptors.

    Science.gov (United States)

    Contreras-Jurado, Constanza; García-Serrano, Laura; Martínez-Fernández, Mónica; Ruiz-Llorente, Lidia; Paramio, Jesus M; Aranda, Ana

    2014-01-01

    Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies.

  5. Lack of resistance development in Bemisia tabaci to Isaria fumosorosea after multiple generations of selection

    Science.gov (United States)

    Gao, Tianni; Wang, Zhaolei; Huang, Yü; Keyhani, Nemat O.; Huang, Zhen

    2017-02-01

    The emergence of insecticide resistant insect pests is of significant concern worldwide. The whitefly, Bemisia tabaci, is an important agricultural pest and has shown incredible resilience developing resistance to a number of chemical pesticides. Entomopathogenic fungi such as Isaria fumosorosea offer an attractive alternative to chemical pesticides for insect control, and this fungus has been shown to be an effective pathogen of B. tabaci. Little is known concerning the potential for the development of resistance to I. fumosorosea by B. tabaci. Five generations of successive survivors of B. tabaci infected by I. fumosorosea were assayed with I. fumosorosea. No significant differences in susceptibility to I. fumosorosea, number of ovarioles, or ovipostioning were seen between any of the generations tested. Effects of I. fumosorosea and cell-free ethyl acetate fractions derived from the fungus on the B. tabaci fat body, ovary, and vitellogenin were also investigated. These data revealed significant deformation and degradation of ovary tissues and associated vitellogenin by the fungal mycelium as well as by cell-free ethyl acetate fungal extracts. These data indicate the lack of the emergence of resistance to I. fumosorosea under the conditions tested and demonstrate invasion of the insect reproductive tissues during fungal infection.

  6. Lack of resistance development in Bemisia tabaci to Isaria fumosorosea after multiple generations of selection

    Science.gov (United States)

    Gao, Tianni; Wang, Zhaolei; Huang, Yü; Keyhani, Nemat O.; Huang, Zhen

    2017-01-01

    The emergence of insecticide resistant insect pests is of significant concern worldwide. The whitefly, Bemisia tabaci, is an important agricultural pest and has shown incredible resilience developing resistance to a number of chemical pesticides. Entomopathogenic fungi such as Isaria fumosorosea offer an attractive alternative to chemical pesticides for insect control, and this fungus has been shown to be an effective pathogen of B. tabaci. Little is known concerning the potential for the development of resistance to I. fumosorosea by B. tabaci. Five generations of successive survivors of B. tabaci infected by I. fumosorosea were assayed with I. fumosorosea. No significant differences in susceptibility to I. fumosorosea, number of ovarioles, or ovipostioning were seen between any of the generations tested. Effects of I. fumosorosea and cell-free ethyl acetate fractions derived from the fungus on the B. tabaci fat body, ovary, and vitellogenin were also investigated. These data revealed significant deformation and degradation of ovary tissues and associated vitellogenin by the fungal mycelium as well as by cell-free ethyl acetate fungal extracts. These data indicate the lack of the emergence of resistance to I. fumosorosea under the conditions tested and demonstrate invasion of the insect reproductive tissues during fungal infection. PMID:28230074

  7. Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes.

    Directory of Open Access Journals (Sweden)

    Konstantin Piatkov

    Full Text Available In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward ("chase", using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT, introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein and an upstream polypeptide (a long-lived reference protein. The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase. Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

  8. Natural Outbreak of BVDV-1d-Induced Mucosal Disease Lacking Intestinal Lesions.

    Science.gov (United States)

    Bianchi, M V; Konradt, G; de Souza, S O; Bassuino, D M; Silveira, S; Mósena, A C S; Canal, C W; Pavarini, S P; Driemeier, D

    2017-03-01

    Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus, which is further divided into subgenotypes (1a-1u and 2a-c). When persistent infection occurs, the calf will be immunotolerant to BVDV and possibly develop mucosal disease. This study describes an outbreak of BVDV-1d-induced mucosal disease lacking intestinal lesions. Eleven calves presented with anorexia, sialorrhea, lameness, recumbency, and death. Three calves were necropsied, showing ulceration of the interdigital skin and the oral and nasal mucosa; linear ulcers in the tongue, esophagus, and rumen; and rounded ulcers in the abomasum. Microscopically, mucosa and skin had superficial necrosis, with single-cell necrosis and vacuolation in epithelial cells, and severe parakeratosis. Immunohistochemistry (IHC) showed BVDV antigen in the cytoplasm of epithelial cells in skin and mucosa. All 11 dead calves were positive upon reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Pestivirus along with another 11 live calves from the herd, which were positive again by RT-PCR and IHC after a 4-week interval. Sequencing of the 5' untranslated region and N-terminal protease showed that viruses from these 22 calves were homologous and of subgenotype BVDV-1d. Cytopathic BVDV was isolated from 8 of 11 dead calves, but only noncytopathic BVDV was isolated from the 11 live animals. The findings indicate that this was an outbreak of mucosal disease caused by BVDV-1d, with high morbidity, and lesions restricted to the upper alimentary system and skin and absent from intestine. Thus, the epidemiological and pathological features in this form of mucosal disease may be similar to vesicular diseases, including foot and mouth disease.

  9. Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene.

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    Vladimir O Murovets

    Full Text Available The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+ inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-. Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.

  10. Upregulation of B7 molecules (CD80 and CD86) and exacerbated eosinophilic pulmonary inflammatory response in mice lacking the IFN-beta gene

    DEFF Research Database (Denmark)

    Matheu, Victor; Treschow, Alexandra; Navikas, Vaidrius

    2003-01-01

    )-sensitized and OVA-challenged mice. RESULTS: OVA-sensitized and OVA-challenged mice with lack of the IFNB gene had more severe pulmonary inflammation with increased lung local response, including IL-4, IL-5, IL-13, IgE, eosinophilia, and goblet cells, than their litter mates (IFN-beta+/-), whereas no differences...... were observed in regard to local levels of IFN-gamma. Moreover, systemic response with IgE production is also enhanced. Lack of IFN-beta also results in significantly higher antigen-specific T cells, with higher levels of IL-4, IL-5, and IL-13, whereas no significant differences in IFN-gamma response...

  11. Lack of the sodium-driven chloride bicarbonate exchanger NCBE impairs visual function in the mouse retina.

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    Gerrit Hilgen

    Full Text Available Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10, a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pH(i and chloride concentration ([Cl(-](i in neurons. Here we show that NCBE is strongly expressed in the retina. As GABA(A receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pH(i regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.

  12. THE LEVELS OF VALUE IN BUSINESS VALUATION. THE DISCOUNT FOR LACK OF MARKETABILITY

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    Szatmary Miclea Camelia

    2012-12-01

    Full Text Available This paper approaches the subject of levels of value used in business valuation and adjustments (discounts and prime that enable professional business valuers to relate these in order to determine the value of a business or ownerships interests in a business. Marketability and control over business decision have a profound impact on the value of an ownership interest. Valuators, regularly, reduce the value of an investment interest if it lacks a ready market. In this paper, I highlight the difference between the marketability and liquidity and, the differences between the the discount for lack of marketability and discount for lack of control. A discount for lack of marketability is closely associated with, but, conceptually, distinct from, the discount for lack of control. I present the characteristics of discount for lack of marketability and the rational of applying it in valuation of minority and majority interest.

  13. Cholesteryl ester hydrolase activity is abolished in HSL-/- macrophages but unchanged in macrophages lacking KIAA1363.

    Science.gov (United States)

    Buchebner, Marlene; Pfeifer, Thomas; Rathke, Nora; Chandak, Prakash G; Lass, Achim; Schreiber, Renate; Kratzer, Adelheid; Zimmermann, Robert; Sattler, Wolfgang; Koefeler, Harald; Fröhlich, Eleonore; Kostner, Gerhard M; Birner-Gruenberger, Ruth; Chiang, Kyle P; Haemmerle, Guenter; Zechner, Rudolf; Levak-Frank, Sanja; Cravatt, Benjamin; Kratky, Dagmar

    2010-10-01

    Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363(-/-) and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL(-/-) mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363(-/-) but unchanged in HSL(-/-) mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL(-/-) macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

  14. A unique hexokinase in Cryptosporidium parvum, an apicomplexan pathogen lacking the Krebs cycle and oxidative phosphorylation.

    Science.gov (United States)

    Yu, Yonglan; Zhang, Haili; Guo, Fengguang; Sun, Mingfei; Zhu, Guan

    2014-09-01

    Cryptosporidium parvum may cause virtually untreatable infections in AIDS patients, and is recently identified as one of the top four diarrheal pathogens in children in developing countries. Cryptosporidium differs from other apicomplexans (e.g., Plasmodium and Toxoplasma) by lacking many metabolic pathways including the Krebs cycle and cytochrome-based respiratory chain, thus relying mainly on glycolysis for ATP production. Here we report the molecular and biochemical characterizations of a hexokinase in C. parvum (CpHK). Our phylogenetic reconstructions indicated that apicomplexan hexokinases including CpHK were highly divergent from those of humans and animals (i.e., at the base of the eukaryotic clade). CpHK displays unique kinetic features that differ from those in mammals and Toxoplasma gondii (TgHK) in the preference towards various hexoses and its capacity to use ATP and other NTPs. CpHK also displays substrate inhibition by ATP. Moreover, 2-deoxy-D-glucose (2DG) could not only inhibit the CpHK activity, but also the parasite growth in vitro at concentrations nontoxic to host cells (IC(50) = 0.54 mM). While the exact action of 2-deoxy-D-glucose on the parasite is subject to further verification, our data suggest that CpHK and the glycolytic pathway may be explored for developing anti-cryptosporidial therapeutics.

  15. Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

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    Camille Benoist

    Full Text Available Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc and induction of the stringent response. This link was confirmed by (pppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX, a potent inducer of stringent response, restored growth of Δsmc under non permissive conditions. We showed that production of (pppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

  16. Lack of Melanopsin Is Associated with Extreme Weight Loss in Mice upon Dietary Challenge.

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    Didem Göz Aytürk

    Full Text Available Metabolic disorders have been established as major risk factors for ocular complications and poor vision. However, little is known about the inverse possibility that ocular disease may cause metabolic dysfunction. To test this hypothesis, we assessed the metabolic consequences of a robust dietary challenge in several mouse models suffering from retinal mutations. To this end, mice null for melanopsin (Opn4-/-, the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs, were subjected to five weeks of a ketogenic diet. These mice lost significantly more weight than wild-type controls or mice lacking rod and cone photoreceptors (Pde6brd1/rd1. Although ipRGCs are critical for proper circadian entrainment, and circadian misalignment has been implicated in metabolic pathology, we observed no differences in entrainment between Opn4-/- and control mice. Additionally, we observed no differences in any tested metabolic parameter between these mouse strains. Further studies are required to establish the mechanism giving rise to this dramatic phenotype observed in melanopsin-null mice. We conclude that the causality between ocular disease and metabolic disorders merits further investigation due to the popularity of diets that rely on the induction of a ketogenic state. Our study is a first step toward understanding retinal pathology as a potential cause of metabolic dysfunction.

  17. Remodeling of the Cervix and Parturition in Mice Lacking the Progesterone Receptor B Isoform1

    Science.gov (United States)

    Yellon, Steven M.; Oshiro, Bryan T.; Chhaya, Tejas Y.; Lechuga, Thomas J.; Dias, Rejane M.; Burns, Alexandra E.; Force, Lindsey; Apostolakis, Ede M.

    2011-01-01

    Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition. PMID:21613631

  18. Mutant mice lacking the p53 C-terminal domain model telomere syndromes.

    Science.gov (United States)

    Simeonova, Iva; Jaber, Sara; Draskovic, Irena; Bardot, Boris; Fang, Ming; Bouarich-Bourimi, Rachida; Lejour, Vincent; Charbonnier, Laure; Soudais, Claire; Bourdon, Jean-Christophe; Huerre, Michel; Londono-Vallejo, Arturo; Toledo, Franck

    2013-06-27

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

  19. Lack of Evidence for Molecular Mimicry in HIV-Infected Subjects.

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    Peter D Burbelo

    Full Text Available Previous studies in HIV patients have reported autoantibodies to several human proteins, including erythropoietin (EPO, interferon-α (IFN-α, interleukin-2 (IL-2, and HLA-DR, as potential mediators of anemia or immunosuppression. The etiology of these autoantibodies has been attributed to molecular mimicry between HIV epitopes and self-proteins. Here, the Luciferase Immunoprecipitation System (LIPS was used to investigate the presence of such autoantibodies in HIV-infected adults. High levels of antibodies to HIV proteins such as capsid (p24, matrix (p17, envelope (gp41, and reverse transcriptase (RT were detected using LIPS in both untreated and anti-retroviral-treated HIV-infected individuals but not in uninfected controls. LIPS readily detected anti-EPO autoantibodies in serum samples from subjects with presumptive pure red cell aplasia but not in any of the samples from HIV-infected or uninfected individuals. Similarly, subjects with HIV lacked autoantibodies to IFN-α, IL-2, HLA-DR and the immunoglobulin lambda light chain; all purported targets of molecular mimicry. While molecular mimicry between pathogen proteins and self-proteins is a commonly proposed mechanism for autoantibody production, the findings presented here indicate such a process is not common in HIV disease.

  20. Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system.

    Science.gov (United States)

    Narita, Yuka; Sato, Keiko; Yukitake, Hideharu; Shoji, Mikio; Nakane, Daisuke; Nagano, Keiji; Yoshimura, Fuminobu; Naito, Mariko; Nakayama, Koji

    2014-10-01

    Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.

  1. Tyrosinase degradation is prevented when EDEM1 lacks the intrinsically disordered region.

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    Marioara B Marin

    Full Text Available EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

  2. Postnatal development of the endocrine pancreas in mice lacking functional GABAB receptors.

    Science.gov (United States)

    Crivello, Martín; Bonaventura, María Marta; Chamson-Reig, Astrid; Arany, Edith; Bettler, Bernhard; Libertun, Carlos; Lux-Lantos, Victoria

    2013-05-15

    Adult mice lacking functional GABAB receptors (GABAB1KO) have glucose metabolism alterations. Since GABAB receptors (GABABRs) are expressed in progenitor cells, we evaluated islet development in GABAB1KO mice. Postnatal day 4 (PND4) and adult, male and female, GABAB1KO, and wild-type littermates (WT) were weighed and euthanized, and serum insulin and glucagon was measured. Pancreatic glucagon and insulin content were assessed, and pancreas insulin, glucagon, PCNA, and GAD65/67 were determined by immunohistochemistry. RNA from PND4 pancreata and adult isolated islets was obtained, and Ins1, Ins2, Gcg, Sst, Ppy, Nes, Pdx1, and Gad1 transcription levels were determined by quantitative PCR. The main results were as follows: 1) insulin content was increased in PND4 GABAB1KO females and in both sexes in adult GABAB1KOs; 2) GABAB1KO females had more clusters (<500 μm(2)) and less islets than WT females; 3) cluster proliferation was decreased at PND4 and increased in adult GABAB1KO mice; 4) increased β-area at the expense of the α-cell area was present in GABAB1KO islets; 5) Ins2, Sst, and Ppy transcription were decreased in PND4 GABAB1KO pancreata, adult GABAB1KO female islets showed increased Ins1, Ins2, and Sst expression, Pdx1 was increased in male and female GABAB1KO islets; and 6) GAD65/67 was increased in adult GABAB1KO pancreata. We demonstrate that several islet parameters are altered in GABAB1KO mice, further pinpointing the importance of GABABRs in islet physiology. Some changes persist from neonatal ages to adulthood (e.g., insulin content in GABAB1KO females), whereas other features are differentially regulated according to age (e.g., Ins2 was reduced in PND4, whereas it was upregulated in adult GABAB1KO females).

  3. Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor.

    Science.gov (United States)

    Fedchenko, Valerii I; Buneeva, Olga A; Kopylov, Arthur T; Veselovsky, Alexander V; Zgoda, Victor G; Medvedev, Alexei E

    2015-01-01

    Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner.

  4. Cells

    Directory of Open Access Journals (Sweden)

    Zhao-Hui Jin

    2012-11-01

    Full Text Available As cancer stem cells (CSCs are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.

  5. Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP2

    OpenAIRE

    Hizaki, Hiroko; Segi, Eri; Sugimoto, Yukihiko; Hirose, Masaya; Saji, Tomomi; Ushikubi, Fumitaka; Matsuoka, Toshiyuki; Noda, Yoichi; Tanaka, Takashi; Yoshida, Nobuaki; Narumiya, Shuh; Ichikawa, Atsushi

    1999-01-01

    Female mice lacking the gene encoding the prostaglandin (PG) E2 receptor subtype EP2 (EP2−/−) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP2−/− females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP2 mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli ...

  6. Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

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    Livas Daniela

    2011-08-01

    Full Text Available Abstract Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA, triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in

  7. Prolongation of chemically-induced methemoglobinemia in mice lacking α-synuclein: A novel pharmacologic and toxicologic phenotype

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    Yien-Ming Kuo

    2015-01-01

    Full Text Available The protein α-synuclein is considered central to the pathogenesis of Parkinson disease (PD on genetic and histopathological grounds. It is widely expressed in fetal life and continues to be highly expressed in adult neural tissues, red blood cells and platelets, while the remainder of adult tissues are reported to have little or no expression. Despite cellular and molecular evidence for a role in neuronal function including synaptic vesicle trafficking, neurotransmitter release, mitochondrial function, lipid metabolism, neurogenesis, neuroprotection, and neuromelanin biosynthesis, mice ablated for the gene encoding α-synuclein (Snca have little or no neurological phenotype. Thus, nearly 20 years of intensive study have yet to reveal conclusively what the normal function of this highly abundant protein is in the nervous system. Interestingly, α-synuclein has also been shown to have enzymatic activity as a ferrireductase capable of reducing Fe+3 to Fe+2. Given its abundant expression in red blood cells, we set out to explore the role of α-synuclein in converting chemically-induced Fe+3 methemoglobin to normal Fe+2 hemoglobin. Initial in vivo experiments with the potent methemoglobin inducer, para-aminopropiophenone and its active metabolite, 4-hydroxy para-aminopropiophenone, demonstrated significantly greater and more prolonged methemoglobinemia in Snca−/− mice compared to Snca+/+ mice. In vitro experiments with red blood cells, however, and in vivo experiments in genetically engineered mouse strains that differ in their α-synuclein expression in various tissues, including the nervous system, red blood cells and liver, revealed that contrary to the initial hypothesis, a lack of expression of α-synuclein in red blood cells did not correlate with higher levels or more prolonged duration of methemoglobinemia. Instead, the greater sensitivity to chemically induced methemoglobinemia correlated with the absence of hepatic

  8. Mice lacking caspase-2 are protected from behavioral changes, but not pathology, in the YAC128 model of Huntington disease

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    Bissada Nagat

    2011-08-01

    Full Text Available Abstract Background Huntington Disease (HD is a neurodegenerative disorder in which caspase activation and cleavage of substrates, including the huntingtin protein, has been invoked as a pathological mechanism. Specific changes in caspase-2 (casp2 activity have been suggested to contribute to the pathogenesis of HD, however unique casp2 cleavage substrates have remained elusive. We thus utilized mice completely lacking casp2 (casp2-/- to examine the role played by casp2 in the progression of HD. This 'substrate agnostic' approach allows us to query the effect of casp2 on HD progression without pre-defining proteolytic substrates of interest. Results YAC128 HD model mice lacking casp2 show protection from well-validated motor and cognitive features of HD, including performance on rotarod, swimming T-maze, pre-pulse inhibition, spontaneous alternation and locomotor tasks. However, the specific pathological features of the YAC128 mice including striatal volume loss and testicular degeneration are unaltered in mice lacking casp2. The application of high-resolution magnetic resonance imaging (MRI techniques validates specific neuropathology in the YAC128 mice that is not altered by ablation of casp2. Conclusions The rescue of behavioral phenotypes in the absence of pathological improvement suggests that different pathways may be operative in the dysfunction of neural circuitry in HD leading to behavioral changes compared to the processes leading to cell death and volume loss. Inhibition of caspase-2 activity may be associated with symptomatic improvement in HD.

  9. Hamster SRD5A3 lacks steroid 5α-reductase activity in vitro.

    Science.gov (United States)

    Chávez, B; Ramos, L; García-Becerra, R; Vilchis, F

    2015-02-01

    According to current knowledge, two steroid 5α-reductases, designated type 1 (SRD5A1) and type 2 (SRD5A2), are present in all species examined to date. These isozymes play a central role in steroid hormone physiology by catalyzing the reduction of 3-keto-4-ene-steroids into more active 5α-reduced derivatives, including the conversion of testosterone (T) to dihydrotestosterone (DHT). A third 5α-reductase (SRD5A3, -type 3), which is overexpressed in hormone-refractory prostate cancer cells, has been identified; however, its enzymatic characteristics are practically unknown. Here, we isolated a cDNA encoding hamster Srd5a3 (hSrd5a3) and performed functional metabolic assays to investigate its biochemical properties. The cloned cDNA encodes a 330 amino acid protein that is 87% identical to the homologous protein in mice and 78% to that in humans. However, hSrd5a3 exhibits low sequence homology with its counterparts hSrd5a1 (19%) and hSrd5a2 (17%). A fusion protein consisting of hSrd5a3 and green fluorescent protein provided evidence for cytoplasmic localization in transfected mammalian cells. Real-time PCR analysis revealed that, Srd5a3 mRNA was present in nearly all hamster tissues, with high expression in the cerebellum, Harderian gland and testis. Functional assays expressing hSrd5a3 cDNA in HEK-293 cells revealed that this isozyme is unable to reduce T into DHT. Further expression assays confirmed that similar to testosterone, progesterone, androstenedione and corticosterone are not reduced by hSrd5a3 or human SRD5A3. Together, these results indicate that hSrd5a3 lacks the catalytic activity to transform 3-keto-4-ene-compounds; therefore 5α-reductase type 3 may not be involved in 5α-reduction of steroids.

  10. 45 CFR 690.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-10-01

    ... Applications and proposals lacking definite plans for involvement of human subjects. Certain types of... knowledge that subjects may be involved within the period of support, but definite plans would not normally... 45 Public Welfare 3 2010-10-01 2010-10-01 false Applications and proposals lacking definite...

  11. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Applications and proposals lacking definite plans for involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement...

  12. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-07-01

    ... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses... and proposals lacking definite plans for involvement of human subjects. Certain types of applications... knowledge that subjects may be involved within the period of support, but definite plans would not...

  13. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-10-01

    ... Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human... 45 Public Welfare 1 2010-10-01 2010-10-01 false Applications and proposals lacking definite plans for involvement of human subjects. 46.118 Section 46.118 Public Welfare DEPARTMENT OF HEALTH AND...

  14. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Applications and proposals lacking definite plans for involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human...

  15. 49 CFR 11.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Applications and proposals lacking definite plans for involvement of human subjects. 11.118 Section 11.118 Transportation Office of the Secretary of Transportation PROTECTION OF HUMAN SUBJECTS § 11.118 Applications and proposals lacking definite plans...

  16. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-07-01

    ... for Protection of Human Research Subjects) § 97.118 Applications and proposals lacking definite plans... 34 Education 1 2010-07-01 2010-07-01 false Applications and proposals lacking definite plans for involvement of human subjects. 97.118 Section 97.118 Education Office of the Secretary, Department...

  17. Broad host range plasmid-based gene transfer system in the cyanobacterium Gloeobacter violaceus which lacks thylakoids

    Institute of Scientific and Technical Information of China (English)

    GUO Haitao; XU Xudong

    2004-01-01

    Gloeobacter violaceus, a cyanobacterium lack of thylakoids, is refractory to genetic manipulations because its cells are enveloped by a thick gelatinous sheath and in colonial form.In this study, a large number of single cells were obtained by repeated pumping with a syringe with the gelatinous sheath removed.And an exogenous broad host range plasmid pKT210 was conjugatively transferred into G.violaceus.Analyses with dot-blot hybridization and restriction mapping showed that the exogenous plasmid pKT210 had been introduced into G.violaceus and stably maintained with no alteration in its structure.pKT210 extracted from G.violaceus exconjugants could be transformed into the mcr- mrr- E.coli strain DH10B but not the mcr+ mrr+ strain DH5α, which suggests that a methylase system may be present in G.violaceus.

  18. Fetal growth retardation and lack of hypotaurine in ezrin knockout mice.

    Directory of Open Access Journals (Sweden)

    Tomohiro Nishimura

    Full Text Available Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/- were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine and taurine were not affected. Lack of hypotaurine in Ez(-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

  19. Lack of developmental redundancy between Unc45 proteins in zebrafish muscle development.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    Full Text Available Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b(-/-; unc45a(-/- mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b(-/- and unc45b(-/-; unc45a(-/- embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.

  20. Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

    Directory of Open Access Journals (Sweden)

    Hales Tim G

    2011-04-01

    Full Text Available Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2 augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

  1. Male-like sexual behavior of female mouse lacking fucose mutarotase

    Directory of Open Access Journals (Sweden)

    Lim Dae-sik

    2010-07-01

    Full Text Available Abstract Background Mutarotases are recently characterized family of enzymes that are involved in the anomeric conversions of monosaccharides. The mammalian fucose mutarotase (FucM was reported in cultured cells to facilitate fucose utilization and incorporation into protein by glycosylation. However, the role of this enzyme in animal has not been elucidated. Results We generated a mutant mouse specifically lacking the fucose mutarotase (FucM gene. The FucM knockout mice displayed an abnormal sexual receptivity with a drastic reduction in lordosis score, although the animals were fertile due to a rare and forced intromission by a typical male. We examined the anteroventral periventricular nucleus (AVPv of the preoptic region in brain and found that the mutant females showed a reduction in tyrosine hydoxylase positive neurons compared to that of a normal female. Furthermore, the mutant females exhibited a masculine behavior, such as mounting to a normal female partner as well as showing a preference to female urine. We found a reduction of fucosylated serum alpha-fetoprotein (AFP in a mutant embryo relative to that of a wild-type embryo. Conclusions The observation that FucM-/- female mouse exhibits a phenotypic similarity to a wild-type male in terms of its sexual behavior appears to be due to the neurodevelopmental changes in preoptic area of mutant brain resembling a wild-type male. Since the previous studies indicate that AFP plays a role in titrating estradiol that are required to consolidate sexual preference of female mice, we speculate that the reduced level of AFP in FucM-/- mouse, presumably resulting from the reduced fucosylation, is responsible for the male-like sexual behavior observed in the FucM knock-out mouse.

  2. Cadmium toxicity to Microcystis aeruginosa PCC 7806 and its microcystin-lacking mutant.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available The adverse effects of microcystin (MC produced by cyanobacteria have drawn considerable attention from the public. Yet it remains unclear whether MC confers any benefits to the cyanobacteria themselves. One suggested function of MC is complexation, which may influence the bioaccumulation and toxicity of trace metals. To test this hypothesis, we examined Cd toxicity to wild-type Microcystis aeruginosa PCC 7806 (WT and its MC-lacking mutant (MT under nutrient-enriched (+NP, phosphorus-limited (-P, and nitrogen-limited (-N conditions. The accumulation of Cd and the biochemical parameters associated with its detoxification [total phosphorus (TP, inorganic polyphosphate (Poly-P, and glutathione (GSH in the cells as well as intra- and extra-cellular carbohydrates] were quantified. Although the -P cyanobacteria accumulated less Cd than their +NP and -N counterparts, the different nutrient-conditioned cyanobacteria were similarly inhibited by similar free ion concentration of Cd in the medium ([Cd2+]F. Such good toxicity predictability of [Cd2+]F was ascribed to the synchronous decrease in the intracellular concentrations of Cd and TP. Nevertheless, Cd toxicity was still determined by the intracellular Cd to phosphorus ratio (Cd/P, in accordance with what has been reported in the literature. On the other hand, the concentrations of TP, Poly-P, and carbohydrates went up, but GSH concentration dropped down with the enhancement of [Cd2+]F, indicating their association with Cd detoxification. Although the inactivation of MC peptide synthetase gene had some nutrient and Cd concentration dependent effects on the parameters above, both cyanobacterial strains showed the same Cd accumulation ability and displayed similar Cd sensitivity. These results suggest that MC cannot affect metal toxicity either by regulating metal accumulation or by altering the detoxification ability of the cyanobacteria. Other possible functions of MC need to be further investigated.

  3. Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6.

    Directory of Open Access Journals (Sweden)

    Chiharu Sogawa

    Full Text Available BACKGROUND: The transporters for dopamine (DAT and norepinephrine (NET are members of the Na+- and Cl--dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT and placenta (NET, both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms. CONCLUSIONS/SIGNIFICANCE: The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.

  4. Lack of Pharmacy Access May Send Some Seniors Back to Hospital

    Science.gov (United States)

    ... medlineplus.gov/news/fullstory_160255.html Lack of Pharmacy Access May Send Some Seniors Back to Hospital ... Aug. 4, 2016 (HealthDay News) -- Limited access to pharmacies may be one reason hospital readmission is more ...

  5. Relation Between Lack of Forgiveness and Depression: The Moderating Effect of Self-Compassion.

    Science.gov (United States)

    Chung, Myung-Sun

    2016-12-01

    Although an association between lack of forgiveness and poor mental health is known, prior studies have reported mixed findings of the relationship between lack of forgiveness and depressive symptoms. In an attempt to explain the strength differences between lack of forgiveness and depressive symptoms, this study examined the moderating effect of self-compassion. A total of 311 Korean teachers (89 men, 222 women; M age = 39.3 year, SD = 9.1) were asked to complete self-report questionnaires, including the Korean versions of the Trait Forgivingness Scale, the Self-Compassion Scale, and the Center for Epidemiologic Studies Depression Scale. Moderated multiple regression was used for analysis, and a buffering interaction of self-compassion was discovered. Specifically, self-compassion moderated the relationship between lack of forgiveness and depression; the relationship was stronger for those low on self-compassion.

  6. Q&A. Does lack of product management impact the users of open source?

    Directory of Open Access Journals (Sweden)

    Paul Young

    2008-05-01

    Full Text Available Most commercial software companies employ product managers to handle the planning and marketing of software products, whereas few open source projects have a product manager. Does lack of product management impact the users of open source?

  7. Terminal Bacteroid Differentiation Is Associated With Variable Morphological Changes in Legume Species Belonging to the Inverted Repeat-Lacking Clade.

    Science.gov (United States)

    Montiel, Jesús; Szűcs, Attila; Boboescu, Iulian Z; Gherman, Vasile D; Kondorosi, Éva; Kereszt, Attila

    2016-03-01

    Medicago and closely related legume species from the inverted repeat-lacking clade (IRLC) impose terminal differentiation onto their bacterial endosymbionts, manifested in genome endoreduplication, cell enlargement, and loss of cell-division capacity. Nodule-specific cysteine-rich (NCR) secreted host peptides are plant effectors of this process. As bacteroids in other IRLC legumes, such as Cicer arietinum and Glycyrrhiza lepidota, were reported not to display features of terminal differentiation, we investigated the fate of bacteroids in species from these genera as well as in four other species representing distinct genera of the phylogenetic tree for this clade. Bacteroids in all tested legumes proved to be larger in size and DNA content than cultured cells; however, the degree of cell elongation was rather variable in the different species. In addition, the reproductive ability of the bacteroids isolated from these legumes was remarkably reduced. In all IRLC species with available sequence data, the existence of NCR genes was found. These results indicate that IRLC legumes provoke terminal differentiation of their endosymbionts with different morphotypes, probably with the help of NCR peptides.

  8. Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

    Directory of Open Access Journals (Sweden)

    E Thomas Danielsen

    2014-06-01

    Full Text Available Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl and the nuclear receptor Knirps (Kni, have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld, controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

  9. Transcriptional Control of Steroid Biosynthesis Genes in the Drosophila Prothoracic Gland by Ventral Veins Lacking and Knirps

    Science.gov (United States)

    Dorry, Elad; Komura-Kawa, Tatsuya; Fujimoto, Yoshinori; Troelsen, Jesper T.; Herder, Rachel; O'Connor, Michael B.; Niwa, Ryusuke; Rewitz, Kim F.

    2014-01-01

    Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development. PMID:24945799

  10. LACK-OF-FIT TESTING FOR LINEAR ERRORS-IN-VARIABLES MODEL%线性测量误差模型的lack-of-fit检验

    Institute of Scientific and Technical Information of China (English)

    宋卫星; 崔恒建

    2002-01-01

    基于正态假设,提出了测量误差模型lack-of-fit检验的一种方法.在原假设下,检验统计量渐近服从χ2分布. 还就该检验的势函数与权函数的选择进行了讨论,同时与文献中相关的方法进行了比较.

  11. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    Science.gov (United States)

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  12. A novel sulindac derivative lacking cyclooxygenase-inhibitory activities suppresses carcinogenesis in the transgenic adenocarcinoma of mouse prostate model.

    Science.gov (United States)

    Zhang, Yong; Zhang, Jinhui; Wang, Lei; Quealy, Emily; Gary, Bernard D; Reynolds, Robert C; Piazza, Gary A; Lü, Junxuan

    2010-07-01

    Nonsteroidal anti-inflammatory drugs including sulindac are well documented to be highly effective for cancer chemoprevention. However, their cyclooxygenase (COX)-inhibitory activities cause severe gastrointestinal, renal, and cardiovascular toxicities, limiting their chronic use. Recent studies suggest that COX-independent mechanisms may be responsible for the chemopreventive benefits of nonsteroidal anti-inflammatory drugs and support the potential for the development of a novel generation of sulindac derivatives lacking COX inhibition for cancer chemoprevention. A prototypic sulindac derivative with a N,N-dimethylammonium substitution called sulindac sulfide amide (SSA) was recently identified to be devoid of COX-inhibitory activity yet displays much more potent tumor cell growth-inhibitory activity in vitro compared with sulindac sulfide. In this study, we investigated the androgen receptor (AR) signaling pathway as a potential target for its COX-independent antineoplastic mechanism and evaluated its chemopreventive efficacy against prostate carcinogenesis using the transgenic adenocarcinoma of mouse prostate model. The results showed that SSA significantly suppressed the growth of human and mouse prostate cancer cells expressing AR in strong association with G(1) arrest, and decreased AR level and AR-dependent transactivation. Dietary SSA consumption dramatically attenuated prostatic growth and suppressed AR-dependent glandular epithelial lesion progression through repressing cell proliferation in the transgenic adenocarcinoma of mouse prostate mice, whereas it did not significantly affect neuroendocrine carcinoma growth. Overall, the results suggest that SSA may be a chemopreventive candidate against prostate glandular epithelial carcinogenesis.

  13. Flower stalk segments of Arabidopsis thaliana ecotype Columbia lack the capacity to grow in response to exogenously applied auxin.

    Science.gov (United States)

    Soga, K; Wakabayashi, K; Hoson, T; Kamisaka, S

    2000-12-01

    Exogenously applied IAA stimulated cell elongation of segments excised from flower stalks of Arabidopsis thaliana ecotype Landsberg erecta (Ler) by increasing the cell wall extensibility, but it did not affect that of ecotype Columbia (Col). Treatment with a low pH buffer solution (pH 4.0) or fusicoccin (FC), a reagent activating H(+)-ATPases, significantly increased the cell wall extensibility and promoted elongation growth of flower stalk segments of both ecotypes, indicating that the flower stalk segments of Col possess the capacity to grow under acidic pH conditions. IAA promoted the proton excretion in segments of Ler but not of Col. On the other hand, FC increased the proton excretion in segments of Col as much as that of Ler. These results suggest that IAA activates the plasma membrane H(+)-ATPases in the segments of Ler but not those of Col, while FC activates them in both ecotypes. Flower stalks of Col may lack the mechanisms of activation by IAA of the plasma membrane H(+)-ATPases.

  14. Mice lacking the synaptic adhesion molecule Neph2/Kirrel3 display moderate hyperactivity and defective novel object preference

    Directory of Open Access Journals (Sweden)

    Su Yeon eChoi

    2015-07-01

    Full Text Available Synaptic adhesion molecules regulate diverse aspects of neuronal synapse development, including synapse specificity, formation, and maturation. Neph2, also known as Kirrel3, is an immunoglobulin superfamily adhesion molecule implicated in intellectual disability, neurocognitive delay associated with Jacobsen syndrome, and autism spectrum disorders. We here report mice lacking Neph2 (Neph2–/– mice display moderate hyperactivity in a familiar but not novel environment and novel object recognition deficit with normal performances in Morris water maze spatial learning and memory, contextual fear conditioning and extinction, and pattern separation tests. These mice show normal levels of anxiety-like behaviors, social interaction, and repetitive behaviors. At the synapse level, Neph2–/– dentate gyrus granule cells exhibit unaltered dendritic spine density and spontaneous excitatory synaptic transmission. These results suggest that Neph2 is important for normal locomotor activity and object recognition memory.

  15. Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

    Science.gov (United States)

    Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

    2013-10-01

    Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters.

  16. Increased callose deposition in plants lacking DYNAMIN-RELATED PROTEIN 2B is dependent upon POWDERY MILDEW RESISTANT 4.

    Science.gov (United States)

    Leslie, Michelle E; Rogers, Sean W; Heese, Antje

    2016-11-01

    Callose deposition within the cell wall is a well-documented plant immune response to pathogenic organisms as well as to pathogen-/microbe- associated molecular patterns (P/MAMPs). However, the molecular mechanisms that modulate pathogen-induced callose deposition are less understood. We reported previously that Arabidopsis plants lacking the vesicle trafficking component DYNAMIN-RELATED PROTEIN 2B (DRP2B) display increased callose deposition in response to the PAMP flg22. Here, we show that increased number of flg22-induced callose deposits in drp2b leaves is fully dependent on the callose synthase POWDERY MILDEW RESISTANT 4 (PMR4). We propose that in addition to functioning in flg22-induced endocytosis of the plant receptor, FLAGELLIN SENSING 2, DRP2B may regulate the trafficking of proteins involved in callose synthesis, such as PMR4, and/or callose degradation.

  17. Predictors of lack of serological response to syphilis treatment in HIV-infected subjects

    Directory of Open Access Journals (Sweden)

    Vincenzo Spagnuolo

    2014-11-01

    Full Text Available Introduction: The aim of this study was to determine factors associated with lack of serological response (LSR to treatment of syphilis among HIV-infected subjects. Materials and Methods: Retrospective, longitudinal study on HIV-infected subjects diagnosed and treated for syphilis and with an assessable serological response between 1 January 2004 and 15 September 2013. LSR was defined as a <4-fold decline of rapid plasma reagin (RPR titer or a failed reversion to nonreactive (if RPR ≤1:4 at diagnosis after one year since treatment. Diagnoses of syphilis were staged in early syphilis (primary, secondary and early latent or late syphilis (tertiary and late latent according to clinical examination and patient's history. Syphilis was classified in new infections [NI: positive RPR and TPHA (Treponema pallidum Haemagglutination assay titers in subjects without previous history of syphilis] or re-infections [ReI: a ≥4-fold increase of RPR titer in subjects previously successfully treated for syphilis]. Syphilis treatment was prescribed according to CDC guidelines. The crude incidence rates (IRs of LSR were calculated per 1000-person months of follow-up (PMFU as the total number of LSR episodes divided by the cumulative time contributed by all subjects (interval time since each syphilis diagnosis and the date of ascertainment of response. Results are described as median (IQR or frequency (%. Results: 565 diagnoses of syphilis with an assessable serological response in 421 patients; 458 (81% were early syphilis, 189 (33% were NI, 376 (67% were ReI. At first, diagnosis of syphilis median age was 41 (36–47 years, 419 (99.5% males, 391 (93% MSM, HIV-infected since 7.7 (3.5–12.9 years, 75 (18% HCV or HBV co-infected, 56 (13% with a previous AIDS diagnosis, 82 (19% antiretroviral treatment naïve, 102 (24% with HIV-RNA ≥50 cp/mL, CD4+=576 (437–749 cells/mm3, nadir CD4+=308 (194–406 cells/mm3. LSRs were observed in 70/565 (12.4% treated syphilis

  18. Competency to stand trial and defendants who lack insight into their mental illness.

    Science.gov (United States)

    Reisner, Andrew D; Piel, Jennifer; Makey, Miller

    2013-01-01

    Forensic evaluators often assess patients who lack insight into their mental illnesses. This lack of insight can have a significant impact on the defendant's ability to make legal strategy decisions that rely on their acceptance of their mental illness. In this article, the relationship between refusing an insanity plea and competency to stand trial will be explored in the context of defendants who lack insight into their mental illness. The authors argue that an adequate competency assessment should take into account the defendant's ability to consider his available pleas rationally. Such evaluations may have the effect of negating the necessity of a Frendak inquiry in those jurisdictions that can impose the insanity defense on defendants.

  19. Development of a lack of appetite item bank for computer-adaptive testing (CAT)

    DEFF Research Database (Denmark)

    Thamsborg, Lise Laurberg Holst; Petersen, Morten Aa; Aaronson, Neil K

    2015-01-01

    measurement precision. The EORTC Quality of Life Group is developing a CAT version of the widely used EORTC QLQ-C30 questionnaire. Here, we report on the development of the lack of appetite CAT. METHODS: The EORTC approach to CAT development comprises four phases: literature search, operationalization, pre......-testing, and field testing. Phases 1-3 are described in this paper. First, a list of items was retrieved from the literature. This was refined, deleting redundant and irrelevant items. Next, new items fitting the "QLQ-C30 item style" were created. These were evaluated by international samples of experts and cancer...... to 12 lack of appetite items. CONCLUSIONS: Phases 1-3 resulted in 12 lack of appetite candidate items. Based on a field testing (phase 4), the psychometric characteristics of the items will be assessed and the final item bank will be generated. This CAT item bank is expected to provide precise...

  20. Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia

    DEFF Research Database (Denmark)

    Ejlerskov, Patrick; Hultberg, Jeanette Göransdotter; Wang, JunYang;

    2015-01-01

    -causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused...... neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.......Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease...

  1. Increased brain-derived neurotrophic factor (BDNF) protein concentrations in mice lacking brain serotonin.

    Science.gov (United States)

    Kronenberg, Golo; Mosienko, Valentina; Gertz, Karen; Alenina, Natalia; Hellweg, Rainer; Klempin, Friederike

    2016-04-01

    The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.

  2. Expansion of the lateral ventricles and ependymal deficits underlie the hydrocephalus evident in mice lacking the transcription factor NFIX.

    Science.gov (United States)

    Vidovic, Diana; Harris, Lachlan; Harvey, Tracey J; Evelyn Heng, Yee Hsieh; Smith, Aaron G; Osinski, Jason; Hughes, James; Thomas, Paul; Gronostajski, Richard M; Bailey, Timothy L; Piper, Michael

    2015-08-01

    Nuclear factor one X (NFIX) has been shown to play a pivotal role during the development of many regions of the brain, including the neocortex, the hippocampus and the cerebellum. Mechanistically, NFIX has been shown to promote neural stem cell differentiation through the activation of astrocyte-specific genes and via the repression of genes central to progenitor cell self-renewal. Interestingly, mice lacking Nfix also exhibit other phenotypes with respect to development of the central nervous system, and whose underlying causes have yet to be determined. Here we examine one of the phenotypes displayed by Nfix(-/-) mice, namely hydrocephalus. Through the examination of embryonic and postnatal Nfix(-/-) mice we reveal that hydrocephalus is first seen at around postnatal day (P) 10 in mice lacking Nfix, and is fully penetrant by P20. Furthermore, we examined the subcommissural organ (SCO), the Sylvian aqueduct and the ependymal layer of the lateral ventricles, regions that when malformed and functionally perturbed have previously been implicated in the development of hydrocephalus. SOX3 is a factor known to regulate SCO development. Although we revealed that NFIX could repress Sox3-promoter-driven transcriptional activity in vitro, SOX3 expression within the SCO was normal within Nfix(-/-) mice, and Nfix mutant mice showed no abnormalities in the structure or function of the SCO. Moreover, these mutant mice exhibited no overt blockage of the Sylvian aqueduct. However, the ependymal layer of the lateral ventricles was frequently absent in Nfix(-/-) mice, suggesting that this phenotype may underlie the development of hydrocephalus within these knockout mice.

  3. Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice.

    Science.gov (United States)

    Pichlmair, Andreas; Buse, Johanna; Jennings, Stephanie; Haller, Otto; Kochs, Georg; Staeheli, Peter

    2004-10-01

    The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.

  4. Mice lacking neutral amino acid transporter B0AT1 (Slc6a19) have elevated levels of FGF21 and GLP-1 and improved glycaemic control

    OpenAIRE

    Yang Jiang; Adam J. Rose; Sijmonsma, Tjeerd P.; Angelika Bröer; Anja Pfenninger; Stephan Herzig; Dieter Schmoll; Stefan Bröer

    2015-01-01

    Objective: Type 2 diabetes arises from insulin resistance of peripheral tissues followed by dysfunction of β-cells in the pancreas due to metabolic stress. Both depletion and supplementation of neutral amino acids have been discussed as strategies to improve insulin sensitivity. Here we characterise mice lacking the intestinal and renal neutral amino acid transporter B0AT1 (Slc6a19) as a model to study the consequences of selective depletion of neutral amino acids. Methods: Metabolic tests...

  5. C60-Fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects

    Directory of Open Access Journals (Sweden)

    Carroll David L

    2006-12-01

    Full Text Available Abstract We have developed a new method of application of C60 to cultured cells that does not require water-solubilization techniques. Normal and malignant cells take-up C60 and the inherent photoluminescence of C60 is detected within multiple cell lines. Treatment of cells with up to 200 μg/ml (200 ppm of C60 does not alter morphology, cytoskeletal organization, cell cycle dynamics nor does it inhibit cell proliferation. Our work shows that pristine C60 is non-toxic to the cells, and suggests that fullerene-based nanocarriers may be used for biomedical applications.

  6. The cytoplasmic location of chicken mx is not the determining factor for its lack of antiviral activity.

    Directory of Open Access Journals (Sweden)

    Camilla T O Benfield

    Full Text Available BACKGROUND: Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV, an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34 gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems. CONCLUSIONS/SIGNIFICANCE: The chicken Mx protein (Asn631 lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does

  7. 25 CFR 170.811 - What happens if lack of funds results in inadequate maintenance?

    Science.gov (United States)

    2010-04-01

    ... maintenance? 170.811 Section 170.811 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN RESERVATION ROADS PROGRAM BIA Road Maintenance § 170.811 What happens if lack of funds results in inadequate maintenance? If BIA determines that an IRR transportation facility is not...

  8. Some remarks on the effects of drugs, lack of sleep and loud noise on human performance.

    NARCIS (Netherlands)

    Sanders, A.F. & A.A. Bunt.

    1971-01-01

    Some literature is reviewed on the effect of some drugs, (amphetamine, hypnotics, alcohol), loud noise and sleep loss in test of time estimation, decision making, long term performance and short term memory. Results are most clear with respect to amphetamine, hypnotics and lack of sleep, in that amp

  9. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  10. Mind Maps to Modify Lack of Attention among Saudi Kindergarten Children

    Science.gov (United States)

    Daghistan, Bulquees Ismail Abdul Majid

    2016-01-01

    This research study aims at investigating the impact of Mind Maps on modifying the lack of attention in Arabic language class among Saudi Kindergarten children. To achieve the goals of this study the researcher used an experimental design with a random sample from AlRae'd Kindergarten's children in Riyadh -Saudi Arabia for the academic year…

  11. Patient demands, lack of reciprocity, and burnout: a five-year longitudinal study about general practitioners.

    NARCIS (Netherlands)

    Bakker, A.B.; Schaufeli, W.B.; Sixma, H.J.; Bosveld, W.; Dierendonck, D. van

    2000-01-01

    This study among a sample of 207 general practitioners (GPs) uses a five-year longitudinal design to test a process model of burnout. On the basis of social exchange and equity theory, it is hypothesized and found that demanding patient contacts produce a lack of reciprocity in the GP-patient rel

  12. Cyanobacterial contribution to the genomes of the plastid-lacking protists

    Directory of Open Access Journals (Sweden)

    Matsuzaki Motomichi

    2009-08-01

    Full Text Available Abstract Background Eukaryotic genes with cyanobacterial ancestry in plastid-lacking protists have been regarded as important evolutionary markers implicating the presence of plastids in the early evolution of eukaryotes. Although recent genomic surveys demonstrated the presence of cyanobacterial and algal ancestry genes in the genomes of plastid-lacking protists, comparative analyses on the origin and distribution of those genes are still limited. Results We identified 12 gene families with cyanobacterial ancestry in the genomes of a taxonomically wide range of plastid-lacking eukaryotes (Phytophthora [Chromalveolata], Naegleria [Excavata], Dictyostelium [Amoebozoa], Saccharomyces and Monosiga [Opisthokonta] using a novel phylogenetic pipeline. The eukaryotic gene clades with cyanobacterial ancestry were mostly composed of genes from bikonts (Archaeplastida, Chromalveolata, Rhizaria and Excavata. We failed to find genes with cyanobacterial ancestry in Saccharomyces and Dictyostelium, except for a photorespiratory enzyme conserved among fungi. Meanwhile, we found several Monosiga genes with cyanobacterial ancestry, which were unrelated to other Opisthokonta genes. Conclusion Our data demonstrate that a considerable number of genes with cyanobacterial ancestry have contributed to the genome composition of the plastid-lacking protists, especially bikonts. The origins of those genes might be due to lateral gene transfer events, or an ancient primary or secondary endosymbiosis before the diversification of bikonts. Our data also show that all genes identified in this study constitute multi-gene families with punctate distribution among eukaryotes, suggesting that the transferred genes could have survived through rounds of gene family expansion and differential reduction.

  13. Relation of Neuroticism and Negative Career Thoughts and Feelings to Lack of Information

    Science.gov (United States)

    Kelly, Kevin R.; Shin, Yun-Jeong

    2009-01-01

    The purpose of this study was to explore correlates of chronic career indecision with multivariate modeling. We examined the effects of neuroticism and negative career thoughts and feelings on lack of information, which is one of the core elements of chronic career indecision. The sample included 310 first-semester students who had entered…

  14. Non-Native Student's Communication Is Affected Due to the Lack of Pragmatic Competence

    Science.gov (United States)

    Latha, V. G.; Rajan, Premalatha

    2012-01-01

    This paper aims at focusing how the lack of pragmatic competence affects student's communication in L2 (Second language) at tertiary level. The city based Indian students learn English which is their second language from 3 years onwards whereas the rural based students learn English only from 6 years onwards. This exposure of the L2 shows the…

  15. Lack of Emotional Support from Parents Early in Life and Alcohol Abuse Later in Life

    Science.gov (United States)

    Shaw, Benjamin A.

    2006-01-01

    The purpose of this study is to examine the association between lacking emotional support from parents early in life and adult alcohol abuse. A series of logistic regression models were run with data collected from a nationally representative sample of over 2,500 adults ages 25-74. The findings reveal a linear relationship between level of…

  16. Do semantic standards lack quality? : a survey among 34 semantic standards

    NARCIS (Netherlands)

    Folmer, Erwin; Oude Luttighuis, Paul; Hillegersberg, van Jos

    2011-01-01

    The adoption of standards to improve interoperability in the automotive, aerospace, shipbuilding and other sectors could save billions. While interoperability standards have been created for a number of industries, problems persist, suggesting a lack of quality of the standards themselves. The issue

  17. Do semantic standards lack quality? A survey among 34 semantic standards

    NARCIS (Netherlands)

    Folmer, E.J.A.; Oude Luttighuis, P.H.W.M.; Hillegersberg, J. van

    2011-01-01

    The adoption of standards to improve interoperability in the automotive, aerospace, shipbuilding and other sectors could save billions. While interoperability standards have been created for a number of industries, problems persist, suggesting a lack of quality of the standards themselves. The issue

  18. Tools to Assess Pain or Lack of Comfort in Dementia: A Content Analysis

    NARCIS (Netherlands)

    Steen, J.T. van der; Sampson, E.L.; Block, L. Van den; Lord, K.; Vankova, H.; Pautex, S.; Vandervoort, A.; Radbruch, L.; Shvartzman, P.; Sacchi, V.; Vet, H.C. de; Noortgate, N.J. Van Den

    2015-01-01

    CONTEXT: There is need for tools to help detect pain or lack of comfort in persons unable to communicate. However, pain and (dis)comfort tools have not been compared, and it is unclear to what extent they discriminate between pain and other possible sources of discomfort, or even if items differ. OB

  19. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

  20. Influence of smoking, alcohol consumption, drug usage, and lack of physical activity on health

    NARCIS (Netherlands)

    Gecková, A.; Pudelsky, M.; Tuinstra, J.; van Dijk, J.P.

    2000-01-01

    In the framework of project "lnequality in health In adolescents", the influence of smoking, alcohol consumption, drug usage, lack of physical activity. and their cumulative influence on health was Investigated in the sample of Slovak adolescents (n = 2616, including 1370 boys, average age 15 years)

  1. The Lack of Political Cartoons in the People's Republic of China.

    Science.gov (United States)

    Schnell, Jim

    Political cartoons do not appear in the government-controlled press in the People's Republic of China. The cartoons that do appear in newspapers are good-natured and lacking in any type of political message. Chinese civilization has a 5,000-year history that is grounded in feudalism and must be considered in any analysis of Chinese society. Since…

  2. Stabilization of oral anticoagulant therapy in hospitalized patients and characteristics associated with lack of stabilization

    NARCIS (Netherlands)

    van den Bemt, PMLA; Joosten, P; Risselada, A; van den Boogaart, MHA; Egberts, ACG; Brouwers, JRBJ

    2000-01-01

    The initiation and stabilization of oral anticoagulant therapy in hospitalized patients in a setting without specialized medical or pharmaceutical advice, was studied. In addition, potential risk factors for lack of stabilization were studied. All patients from three wards (orthopaedic surgery, gene

  3. Private Pre-University Education in Romania: Mixing Control with Lack of Strategy

    Science.gov (United States)

    Stanus, Cristina

    2014-01-01

    This paper approaches private provision of pre-university education in Romania, exploring available data on the sector's size and main characteristics and evaluating the extent to which the current regulatory framework enables positive effects in terms of freedom of choice, quality, equity, and social cohesion. The paper argues that the lack of a…

  4. Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42

    DEFF Research Database (Denmark)

    Pleines, Irina; Dütting, Sebastian; Cherpokova, Deya;

    2013-01-01

    normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were...

  5. Lack of diffuseness in wave propagation in lightweight joist-floors

    DEFF Research Database (Denmark)

    Brunskog, Jonas; Chung, Hyuck

    2007-01-01

    . The structures under consideration are rib-reinforced plate structures. These may be a lightweight floor/joist structure in a building or hull of a ship. The diffuseness or rather the lack of diffuseness, in such 'ribbed-plate' structures is examined. The amount of power transported at an angle is calculated...... by means of the structural intensity in that direction...

  6. 22 CFR 225.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-04-01

    ... plans for involvement of human subjects. 225.118 Section 225.118 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT PROTECTION OF HUMAN SUBJECTS § 225.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants,...

  7. 15 CFR 27.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-01-01

    ... definite plans for involvement of human subjects. 27.118 Section 27.118 Commerce and Foreign Trade Office of the Secretary of Commerce PROTECTION OF HUMAN SUBJECTS § 27.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants,...

  8. 28 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-07-01

    ... definite plans for involvement of human subjects. 46.118 Section 46.118 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROTECTION OF HUMAN SUBJECTS § 46.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants,...

  9. 14 CFR 1230.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-01-01

    ... plans for involvement of human subjects. 1230.118 Section 1230.118 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION PROTECTION OF HUMAN SUBJECTS § 1230.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for...

  10. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Science.gov (United States)

    2010-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense....118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types... agencies with the knowledge that subjects may be involved within the period of support, but definite...

  11. Who Lacks Support and Why? An Examination of Mothers' Personal Safety Nets

    Science.gov (United States)

    Harknett, Kristen S.; Hartnett, Caroline Sten

    2011-01-01

    We use data from the Fragile Families and Child Wellbeing Study (N = 12,140 person-waves) to identify characteristics associated with mothers' having or lacking "personal safety net" support from family and friends. We focus on characteristics that are likely to increase the importance of having support available but may also interfere with the…

  12. Lack of stimulation of 24-hour growth hormone release by hypocaloric diet in obesity

    DEFF Research Database (Denmark)

    Rasmussen, M H; Juul, A; Kjems, L L

    1995-01-01

    . This suggests a reversible defect in GH release, rather than a persistent preexisting disorder. It is hypothesized that enhanced bioavailability of IGF-I, acting in concert with elevated proinsulin and insulin levels, may account for the lack of stimulation of 24-hr GH release by the hypocaloric diet in obese...... and perpetuate the obese state....

  13. Special Deliveries: Certified Nurse-Midwifery Programs Lacking in New England

    Science.gov (United States)

    Franzosa, Alyssa

    2012-01-01

    With Boston serving as a hub of both educational and medical excellence, it's no wonder that New England has a high reputation to uphold in both of these areas. However, Boston and the rest of the region lack a specific degree program that is putting New England below the radars of potential midwives. Certified nurse-midwifery is a popular field…

  14. Analysis of complete mitochondrial genomes from extinct and extant rhinoceroses reveals lack of phylogenetic resolution

    DEFF Research Database (Denmark)

    Willerslev, Eske; Gilbert, Tom; Binladen, Jonas

    2009-01-01

    genes is highly diffuse, with mixed topological support from different genes. Furthermore, the choice of outgroup (horse vs tapir) has considerable effect on reconstruction of the phylogeny. The lack of resolution is suggestive of a hard polytomy at the base of crown-group Rhinocerotidae...

  15. Regional Fluctuation in the Functional Consequence of LINE-1 Insertion in the Mitf Gene: The Black Spotting Phenotype Arisen from the Mitfmi-bw Mouse Lacking Melanocytes.

    Science.gov (United States)

    Takeda, Kazuhisa; Hozumi, Hiroki; Ohba, Koji; Yamamoto, Hiroaki; Shibahara, Shigeki

    2016-01-01

    Microphthalmia-associated transcription factor (Mitf) is a key regulator for differentiation of melanoblasts, precursors to melanocytes. The mouse homozygous for the black-eyed white (Mitfmi-bw) allele is characterized by the white-coat color and deafness with black eyes due to the lack of melanocytes. The Mitfmi-bw allele carries LINE-1, a retrotransposable element, which results in the Mitf deficiency. Here, we have established the black spotting mouse that was spontaneously arisen from the homozygous Mitfmi-bw mouse lacking melanocytes. The black spotting mouse shows multiple black patches on the white coat, with age-related graying. Importantly, each black patch also contains hair follicles lacking melanocytes, whereas the white-coat area completely lacks melanocytes. RT-PCR analyses of the pigmented patches confirmed that the LINE-1 insertion is retained in the Mitf gene of the black spotting mouse, thereby excluding the possibility of the somatic reversion of the Mitfmi-bw allele. The immunohistochemical analysis revealed that the staining intensity for beta-catenin was noticeably lower in hair follicles lacking melanocytes of the homozygous Mitfmi-bw mouse and the black spotting mouse, compared to the control mouse. In contrast, the staining intensity for beta-catenin and cyclin D1 was higher in keratinocytes of the black spotting mouse, compared to keratinocytes of the control mouse and the Mitfmi-bw mouse. Moreover, the keratinocyte layer appears thicker in the Mitfmi-bw mouse, with the overexpression of Ki-67, a marker for cell proliferation. We also show that the presumptive black spots are formed by embryonic day 15.5. Thus, the black spotting mouse provides the unique model to explore the molecular basis for the survival and death of developing melanoblasts and melanocyte stem cells in the epidermis. These results indicate that follicular melanocytes are responsible for maintaining the epidermal homeostasis; namely, the present study has provided

  16. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections

    Energy Technology Data Exchange (ETDEWEB)

    De Benedetti, A.; Pytel, B.A.; Baglioni, C.

    1987-02-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested.

  17. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections.

    Science.gov (United States)

    De Benedetti, A; Pytel, B A; Baglioni, C

    1987-01-01

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested. Images PMID:2433688

  18. Lack of TCRalphabeta+ CD8+ and TCRgammadelta+ lymphocytes ameliorates LPS induced orchitis in mice--preliminary histological observations.

    Science.gov (United States)

    Sliwa, Leopold; Macura, Barbara; Majewska-Szczepanik, Monika; Szczepanik, Marian

    2014-01-01

    The inflammation of the reproductive system can affect reproduction causing partial or complete infertility. It is well known that lipopolysaccharide (LPS) triggers an inflammatory response in the whole organism, including immunologically privileged organs, e.g. the testicles. Adult male TCRalpha-/-, TCRdelta-/-, CD1d-/- and beta2m-/- on B10.PL (H-2(u)) and B10.PL control mice were intraperitonealy (i.p.) injected with lipopolysaccharide (LPS). The animals were killed 24h and 10 days post LPS treatment and their gonads were prepared for microscopic examination. Histological changes in the testes after LPS injection were found only in control B10PL and CD1d-/- mice. The experiments revealed disturbances in Leydig's glands structure, blood vessel dilatation in the interstitial tissue as well as degeneration of seminal tubule epithelium, disruption ofspermatogenesis and subsequent decrease of sperm cell number in the tubule lumen. These changes were noticed mainly 10 days after LPS treatment. Lack of either TCRalphabeta+ CD8+ or TCRgammadelta+ lymphocytes diminishes the response of testicular macrophages to LPS whereas the absence of CD1d-dependent NKT cells does not affect macrophage reactivity.

  19. Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

    Directory of Open Access Journals (Sweden)

    Ali Vural

    Full Text Available In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1, and Regulator of G-protein Signaling 19 (RGS19. As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-, Gpsm1(-/-, or Rgs19(-/- mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-, and Gpsm1(-/- macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

  20. Impaired Lymphoid Organ Development in Mice Lacking the Heparan Sulfate Modifying Enzyme Glucuronyl C5-Epimerase

    NARCIS (Netherlands)

    R.M. Reijmers; M.F.R. Vondenhoff; R. Roozendaal; A. Kuil; J.P. Li; M. Spaargaren; S.T. Pals; R.E. Mebius

    2010-01-01

    The development of lymphoid organs depends on cross talk between hematopoietic cells and mesenchymal stromal cells and on vascularization of the lymphoid primordia. These processes are orchestrated by cytokines, chemokines, and angiogenic factors that require tight spatiotemporal regulation. Heparan

  1. The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

    Science.gov (United States)

    Tamaki, K; Huang, X L; Mizutani, M; Yamamoto, T; Katsumata, R; Uchihi, R; Katsumata, Y; Jeffreys, A J

    1999-07-01

    Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.

  2. Development of speech services for people with cleft palate in Thailand: lack of professionals.

    Science.gov (United States)

    Prathanee, Benjamas

    2012-11-01

    Cleft lip/palate is one of the most common birth defects and has a high incidence in Thailand. Most children with cleft still have social stigma from speech and language defects after surgical treatment. Speech and language therapies are required at an early age and require long-term care until teenager or adult. Unfortunately, there are insufficient speech services for cleft because of a lack of qualified speech and language pathologists in Thailand. Development consisted of two remedy modalities of bottom-up and top-down models, Community-Based Speech Therapy Model for people with Cleft Lip Cleft Palate including networking and standard assessments of both subjective and objective measurements. That might be the best and most suitable way to solve problems of lacking speech services in Thailand or developing countries which have similar contexts.

  3. Lack of credibility in food markets - driving medium quality food out of the market

    DEFF Research Database (Denmark)

    Christensen, Jan; Graversen, Jesper Tranbjerg

    Some food markets are dominated by high quality and standard quality segments, whereas me-dium quality products are almost absent. A modeling framework with asymmetric information regard-ing true quality of the products and the resulting lack of consumer confidence is presented. Uncer-tainty rega......Some food markets are dominated by high quality and standard quality segments, whereas me-dium quality products are almost absent. A modeling framework with asymmetric information regard-ing true quality of the products and the resulting lack of consumer confidence is presented. Uncer......-tainty regarding the quality of alleged medium quality products provides certain consumer groups to divert consumptions away from medium quality to either standard or high quality products. These countervailing incentives explain the missing medium quality products. Empirical examples are given to motivate...

  4. Lacking Community Out-Reach of Chinese Mining Investors in the Arctic

    DEFF Research Database (Denmark)

    Zeuthen, Jesper Willaing

    2016-01-01

    Lacking Community Out-Reach of Chinese Mining Investors in the Arctic Despite China’s bad reputation as a mining investor, in a context of dramatically falling mineral prices, Chinese investments seem to be needed in order to realize most new mining projects across the globe. In Greenland...... and Arctic Canada, potential Chinese investors have been met with even more suspicion than elsewhere. National governments are worried about what state owned Chinese investors will mean for their control over national resources while local governments and the public fear what Chinese investors will mean...... for labour conditions and local environment. They fear a lack of social corporate responsibility (CSR) from Chinese investors. This paper assumes that the possible interest in Arctic mineral resources by the Chinese state combined with a strong demand from Greenland and Canada would make the Arctic a most...

  5. NADP+-dependent glutamate dehydrogenase activity is impaired in mutants of Saccharomyces cerevisiae that lack aconitase.

    Science.gov (United States)

    González, A; Rodríguez, L; Olivera, H; Soberón, M

    1985-10-01

    A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five- to tenfold less NADP+-dependent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible. When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased. Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content. We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.

  6. Nonclinical challenges of Indian dermatology - cities vs. villages, poverty, and lack of awareness.

    Science.gov (United States)

    Verma, Shyam

    2007-10-01

    As India shines in its enviable achievements, one after the other in rapid succession, it also invites criticism from visitors for what they witness. It is imperative to delve deeper to fathom the realities of the predominantly rural India with its poverty and lack of awareness which are stumbling blocks and are major pitfalls in the practice of dermatology. The urban-rural divide, the phenomenon of migration, poverty, lack of awareness all affect practice of dermatology in this country. These are battles that have no ready answers. However a deeper understanding of the ground reality is a worthwhile exercise for the increasing tribe of international dermatologists who like to explore "cross cultural dermatology".

  7. Lack of action of exogenously administered T3 on the fetal rat brain despite expression of the monocarboxylate transporter 8.

    Science.gov (United States)

    Grijota-Martínez, Carmen; Díez, Diego; Morreale de Escobar, Gabriella; Bernal, Juan; Morte, Beatriz

    2011-04-01

    Mutations of the monocarboxylate transporter 8 gene (MCT8, SLC16A2) cause the Allan-Herndon-Dudley syndrome, an X-linked syndrome of severe intellectual deficit and neurological impairment. Mct8 transports thyroid hormones (T4 and T3), and the Allan-Herndon-Dudley syndrome is likely caused by lack of T3 transport to neurons during critical periods of fetal brain development. To evaluate the role of Mct8 in thyroid hormone action in the fetal brain we administered T4 or T3 to thyroidectomized pregnant dams treated with methyl-mercapto-imidazol to produce maternal and fetal hypothyroidism. Gene expression was then measured in the fetal cerebral cortex. T4 increased Camk4, Sema3c, and Slc7a3 expression, but T3 was without effect. To investigate the cause for the lack of T3 action we analyzed the expression of organic anion transport polypeptide (Oatp14, Slco1c1), a T4 transporter, and Mct8 (Slc16a2), a T4 and T3 transporter, by confocal microscopy. Both proteins were present in the brain capillaries forming the blood-brain barrier and in the epithelial cells of the choroid plexus forming the blood-cerebrospinal fluid barrier. It is concluded that T4 from the maternal compartment influences gene expression in the fetal cerebral cortex, possibly after transport via organic anion transporter polypeptide and/or Mct8, and conversion to T3 in the astrocytes. On the other hand, T3 does not reach the target neurons despite the presence of Mct8. The data indicate that T4, through local deiodination, provides most T3 in the fetal rat brain. The role of Mct8 as a T3 transporter in the fetal rat brain is therefore uncertain.

  8. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

    Directory of Open Access Journals (Sweden)

    Moar William J

    2005-06-01

    Full Text Available Abstract Background Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. Results Suppression Subtractive Hybridization (SSH was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4. A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1 were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3, the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. Conclusion We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.

  9. Lack of Bystander Effects From High LET Radiation For Early Cytogenetic Endpoints.

    Energy Technology Data Exchange (ETDEWEB)

    Groesser, Torsten; Cooper, Brian; Rydberg, Bjorn

    2008-05-07

    The aim of this work was to study radiation-induced bystander effects for early cytogenetic end points in various cell lines using the medium transfer technique after exposure to high- and low-LET radiation. Cells were exposed to 20 MeV/ nucleon nitrogen ions, 968 MeV/nucleon iron ions, or 575 MeV/nucleon iron ions followed by transfer of the conditioned medium from the irradiated cells to unirradiated test cells. The effects studied included DNA double-strand break induction, {gamma}-H2AX focus formation, induction of chromatid breaks in prematurely condensed chromosomes, and micronucleus formation using DNA repair-proficient and -deficient hamster and human cell lines (xrs6, V79, SW48, MO59K and MO59J). Cell survival was also measured in SW48 bystander cells using X rays. Although it was occasionally possible to detect an increase in chromatid break levels using nitrogen ions and to see a higher number of {gamma}-H2AX foci using nitrogen and iron ions in xrs6 bystander cells in single experiments, the results were not reproducible. After we pooled all the data, we could not verify a significant bystander effect for any of these end points. Also, we did not detect a significant bystander effect for DSB induction or micronucleus formation in these cell lines or for clonogenic survival in SW48 cells. The data suggest that DNA damage and cytogenetic changes are not induced in bystander cells. In contrast, data in the literature show pronounced bystander effects in a variety of cell lines, including clonogenic survival in SW48 cells and induction of chromatid breaks and micronuclei in hamster cells. To reconcile these conflicting data, it is possible that the epigenetic status of the specific cell line or the precise culture conditions and medium supplements, such as serum, may be critical for inducing bystander effects.

  10. Patient reported outcomes (PROs) in clinical trials: is 'in-trial' guidance lacking? a systematic review.

    OpenAIRE

    Kyte, DG; Draper, H; Ives, J.; Liles, C; Gheorghe, A.; Calvert, M

    2013-01-01

    BACKGROUND: Patient reported outcomes (PROs) are increasingly assessed in clinical trials, and guidelines are available to inform the design and reporting of such trials. However, researchers involved in PRO data collection report that specific guidance on 'in-trial' activity (recruitment, data collection and data inputting) and the management of 'concerning' PRO data (i.e., data which raises concern for the well-being of the trial participant) appears to be lacking. The purpose of this revie...

  11. Genomic Features of Environmental and Clinical Vibrio parahaemolyticus Isolates Lacking Recognized Virulence Factors Are Dissimilar

    OpenAIRE

    Ronholm, J.; Petronella, N.; Chew Leung, C.; Pightling, A. W.; Banerjee, S. K.

    2016-01-01

    Vibrio parahaemolyticus is a bacterial pathogen that can cause illness after the consumption or handling of contaminated seafood. The primary virulence factors associated with V. parahaemolyticus illness are thermostable direct hemolysin (TDH) and Tdh-related hemolysin (TRH). However, clinical strains lacking tdh and trh have recently been isolated, and these clinical isolates are poorly understood. To help understand the emergence of clinical tdh- and trh-negative isolates, a genomic approac...

  12. Synergistic Impairment of Glucose Homeostasis in ob/ob Mice Lacking Functional Serotonin 2C Receptors

    OpenAIRE

    Wade, Jennifer M.; Juneja, Punita; Mackay, Adrienne W.; Graham, James; Havel, Peter J.; Tecott, Laurence H.; Goulding, Evan H.

    2007-01-01

    To investigate how serotonin and leptin interact in the regulation of energy balance and glucose homeostasis, we generated a genetic mouse model, the OB2C mouse, which lacks functional serotonin 2C receptors and the adipocyte hormone leptin. The OB2C mice exhibited a dramatic diabetes phenotype, evidenced by a synergistic increase in serum glucose levels and water intake. The severity of the animals’ diabetes phenotype would not have been predicted from the phenotypic characterization of mice...

  13. Financial Services for the Poor: Lack of Personal Identification Documents Impedes Access

    OpenAIRE

    Nicola Jentzsch

    2009-01-01

    Without a birth certificate, no identity card can be issued and without identity card, there is no access to formal financial services. This link seems to be trivial in industrialized countries, where the ability of the individual to participate in economic life is rarely hindered by a lack of identification. In many developing countries, however, access to financial services is often denied, because potential customers cannot be identified based upon official identity documents-a basic due d...

  14. Migrant Workers' Lack of Cultural Rights and Interests and Government Safeguard Measures

    OpenAIRE

    Zhang, Qiang; Yin, Huan-ju; Wang, Xue-Yan

    2011-01-01

    This paper analyses the status quo of migrant workers' lack of cultural rights and interests as follows: the holistic cultural life of migrant workers is scant and monotonous; the cultural facilities prodded by the majority of enterprises are critically short; the cultural life of migrant workers has the characteristic of closeness to some extent; the holistic cultural consuming capacity of migrant workers is very low, and they are not satisfied with their own cultural life. this paper puts f...

  15. Mice lacking functional STAT1 are highly susceptible to lethal infection with Lassa virus.

    Science.gov (United States)

    Yun, Nadezhda E; Seregin, Alexey V; Walker, David H; Popov, Vsevolod L; Walker, Aida G; Smith, Jeanon N; Miller, Milagros; de la Torre, Juan C; Smith, Jennifer K; Borisevich, Viktoriya; Fair, Joseph N; Wauquier, Nadia; Grant, Donald S; Bockarie, Bayon; Bente, Dennis; Paessler, Slobodan

    2013-10-01

    Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.

  16. Lack of Young Subsidence in the East Tibetan Foreland: Implications for Crustal Thickening Processes at Depth

    Science.gov (United States)

    Royden, L. H.; Burchfiel, B. C.

    2008-12-01

    The Wenchuan earthquake of May 12, 2008 occurred on a west-dipping reverse fault (with a pronounced right-slip component) located along the steep eastern edge of the Tibetan plateau. It has been suggested that thrust faulting here may not be indicative of large-scale shortening and thickening of the upper crust, but may rather be an expression of vertical uplift of the upper crust, with minor shortening. This interpretation is compatible with the lack of young flexural subsidence in the Sichuan foreland provided that the flexurally competent layers of the Sichuan foreland lithosphere are loaded from below, or internally, by thickening crustal domains deep within the mid or lower crust of the eastern plateau, rather than from above, by emplacement of thrust sheets at shallow crustal levels onto the flexurally competent layer of the foreland. This interpretation reconciles gravity anomalies across the plateau margin, the young age of the high topography of eastern Tibet, and the old age of the Sichuan basin with the lack of Cenozoic flexural subsidence in the Sichuan foreland. A similar lack of asymmetric foreland subsidence is also present along the northeastern margin of the Tibetan plateau where it abuts the (southeastern) Tarim Basin, suggesting that a similar mechanism may operate here.

  17. Lack of genetic polymorphism among peregrine falcons Falco peregrinus of Fiji

    Science.gov (United States)

    Talbot, Sandra; Palmer, A.G.; Sage, G.K.; Sonsthagen, Sarah A.; Swem, T.; Brimm, D.J.

    2014-01-01

    We compared levels of genetic diversity and isolation among peregrine falcons Falco peregrinus from two South Pacific island complexes (Fiji and Vanuatu: F. p. nesiotes), relative to other island and mainland populations. Fragment data from 12 microsatellite loci and sequence information from the control region of the mitochondrial DNA indicated levels of genetic variation in the South Pacific populations were lower than other island and mainland populations. Indeed, diversity varied from extremely low (Vanuatu) to completely absent (Fiji). We find little support for a hypothesis that populations on Fiji or Vanuatu were colonized via Australia. The complete lack of polymorphism in peregrine falcons of Fiji is remarkable, and to our knowledge has not been observed in a natural avian population. This lack of polymorphism, and the inability to test for decrease in polymorphism using museum samples, precludes testing whether the lack of genetic diversity in the population on Fiji is due to a recent bottleneck, or sustained isolation over evolutionary time. Increased fertility in eggs of Fiji peregrines upon outbreeding with males from other areas is consistent with inbreeding depression within a population typified by heterozygote deficiency.

  18. ANALYSIS OF A NETWORK OF COOPERATION IN MISIONES, ARGENTINA: BENEFITS AND LACKS FOR LOCAL SUSTAINABLE DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Juan Carlos, Michalus

    2012-01-01

    Full Text Available We present a case study of a network of existing cooperation in the province of Misiones, Argentina, composed of Small and Medium Enterprises (SMEs Elaboration of black tea and a group of SMEs from sawn wood, formed from the particular initiative of the tea entrepreneur. The aim is to identify the benefits of working in cooperation and, in turn, highlight the lacks when these processes are not performed in a planned and sustainable local oriented development. The case study that is exposed is a unique, contemporary data survey with establishments belonging to the cooperative network, interviews with entrepreneurs and managers of production, which led to evidence the benefits of cooperation, which include: use of waste, generation of trust between employers and contribution to environmental stewardship. There were also major shortcomings that prevent greater profit derived from the lack of an appropriate methodological instrument to implement this type of cooperative enterprises, among which stands out: poor use of idle resources, lack of process improvements through innovations joint, non-participation of major players and systematic orientation towards the local sustainable development.

  19. Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity.

    Science.gov (United States)

    Payan, P; Edeyer, A; de Pontual, H; Borelli, G; Boeuf, G; Mayer-Gostan, N

    1999-07-01

    Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.

  20. Lack of habituation of evoked visual potentials in analytic information processing style: evidence in healthy subjects.

    Science.gov (United States)

    Buonfiglio, Marzia; Toscano, M; Puledda, F; Avanzini, G; Di Clemente, L; Di Sabato, F; Di Piero, V

    2015-03-01

    Habituation is considered one of the most basic mechanisms of learning. Habituation deficit to several sensory stimulations has been defined as a trait of migraine brain and also observed in other disorders. On the other hand, analytic information processing style is characterized by the habit of continually evaluating stimuli and it has been associated with migraine. We investigated a possible correlation between lack of habituation of evoked visual potentials and analytic cognitive style in healthy subjects. According to Sternberg-Wagner self-assessment inventory, 15 healthy volunteers (HV) with high analytic score and 15 HV with high global score were recruited. Both groups underwent visual evoked potentials recordings after psychological evaluation. We observed significant lack of habituation in analytical individuals compared to global group. In conclusion, a reduced habituation of visual evoked potentials has been observed in analytic subjects. Our results suggest that further research should be undertaken regarding the relationship between analytic cognitive style and lack of habituation in both physiological and pathophysiological conditions.

  1. Lack of viral control and development of cART escape mutations in macaques after bone marrow transplantation

    Science.gov (United States)

    PETERSON, Christopher W.; HAWORTH, Kevin G.; POLACINO, Patricia; HUANG, Meei-Li; SYKES, Craig; OBENZA, Willimark M.; REPETTO, Andrea C.; KASHUBA, Angela; BUMGARNER, Roger; DeROSA, Stephen C.; WOOLFREY, Ann E.; JEROME, Keith R.; MULLINS, James I.; HU, Shiu-Lok; KIEM, Hans-Peter

    2015-01-01

    Objective We have previously demonstrated robust control of simian/human immunodeficiency virus (SHIV1157-ipd3N4) viremia following administration of combination antiretroviral therapy (cART) in pigtailed macaques. Here, we sought to determine the safety of hematopoietic stem cell transplantation (HSCT) in cART-suppressed and unsuppressed animals. Design We compared disease progression in animals challenged with SHIV 100 days post-transplant (PT), to controls that underwent transplant following SHIV challenge and stable, cART-dependent viral suppression. Methods SHIV viral load, combination antiretroviral therapy (cART) levels, and anti-SHIV antibodies were measured longitudinally from plasma/serum from each animal. Flow cytometry was used to assess T-cell subset frequencies in peripheral blood and gastrointestinal tract (GI). Deep sequencing was used to identify cART resistance mutations. Results In control animals, virus challenge induced transient peak viremia, viral set point, and durable suppression by cART. Subsequent HSCT was not associated with adverse events in these animals. PT animals were challenged during acute recovery following HSCT, and displayed sustained peak viremia and cART resistance. Although PT animals had comparable plasma levels of antiretroviral drugs and showed no evidence of enhanced infection of myeloid subsets in the periphery, they exhibited a drastic reduction in virus-specific antibody production and decreased T-cell counts. Conclusions These results suggest that virus challenge prior to complete transplant recovery impairs viral control and may promote drug resistance. These findings may also have implications for scheduled treatment interruption (STI) studies in patients on cART during post-HSCT recovery: premature STI could similarly result in lack of viral control and cART resistance. PMID:26372270

  2. Transcriptomic analysis reveals how a lack of potassium ions increases Sulfolobus acidocaldarius sensitivity to pH changes.

    Science.gov (United States)

    Buetti-Dinh, Antoine; Dethlefsen, Olga; Friedman, Ran; Dopson, Mark

    2016-08-01

    Extremely acidophilic microorganisms (optimum growth pH of ≤3) maintain a near neutral cytoplasmic pH via several homeostatic mechanisms, including an inside positive membrane potential created by potassium ions. Transcriptomic responses to pH stress in the thermoacidophilic archaeon, Sulfolobus acidocaldarius were investigated by growing cells without added sodium and/or potassium ions at both optimal and sub-optimal pH. Culturing the cells in the absence of added sodium or potassium ions resulted in a reduced growth rate compared to full-salt conditions as well as 43 and 75 significantly different RNA transcript ratios, respectively. Differentially expressed RNA transcripts during growth in the absence of added sodium ions included genes coding for permeases, a sodium/proline transporter and electron transport proteins. In contrast, culturing without added potassium ions resulted in higher RNA transcripts for similar genes as a lack of sodium ions plus genes related to spermidine that has a general role in response to stress and a decarboxylase that potentially consumes protons. The greatest RNA transcript response occurred when S. acidocaldarius cells were grown in the absence of potassium and/or sodium at a sub-optimal pH. These adaptations included those listed above plus osmoregulated glucans and mechanosensitive channels that have previously been shown to respond to osmotic stress. In addition, data analyses revealed two co-expressed IclR family transcriptional regulator genes with a previously unknown role in the S. acidocaldarius pH stress response. Our study provides additional evidence towards the importance of potassium in acidophile growth at acidic pH.

  3. Erythropoietic defect associated with reduced cell proliferation in mice lacking the 26S proteasome shuttling factor Rad23b

    NARCIS (Netherlands)

    S. Bergink (Steven); A.F. Theil (Arjan); W. Toussaint (Wendy); I.M. de Cuyper (Iris); D.I. Kulu (Divine); T. Clapes (Thomas); R. van der Linden (Reinier); J.A.A. Demmers (Jeroen); E.P. Mul (Eric); F.P. van Alphen (Floris); J.A. Marteijn (Jurgen); T. van Gent (Teus); A. Maas (Alex); C. Robin (Catherine); J.N.J. Philipsen (Sjaak); W. Vermeulen (Wim); J.R. Mitchell (James); L. Gutiérrez (Laura)

    2013-01-01

    textabstractRad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely

  4. Erythropoietic Defect Associated with Reduced Cell Proliferation in Mice Lacking the 26S Proteasome Shuttling Factor Rad23b

    NARCIS (Netherlands)

    Bergink, Steven; Theil, Arjan F.; Toussaint, Wendy; De Cuyper, Iris M.; Kulu, Divine I.; Clapes, Thomas; van der Linden, Reinier; Demmers, Jeroen A.; Mul, Eric P.; van Alphen, Floris P.; Marteijn, Jurgen A.; van Gent, Teus; Maas, Alex; Robin, Catherine; Philipsen, Sjaak; Vermeulen, Wim; Mitchell, James R.; Gutierrez, Laura

    2013-01-01

    Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the r

  5. Normal Proliferation and Tumorigenesis but Impaired Pancreatic Function in Mice Lacking the Cell Cycle Regulator Sei1

    OpenAIRE

    Fernandez-Marcos, Pablo J.; Cristina Pantoja; Agueda Gonzalez-Rodriguez; Nicholas Martin; Flores, Juana M.; Valverde, Angela M.; Eiji Hara; Manuel Serrano

    2010-01-01

    Sei1 is a positive regulator of proliferation that promotes the assembly of Cdk4-cyclin D complexes and enhances the transcriptional activity of E2f1. The potential oncogenic role of Sei1 is further suggested by its overexpression in various types of human cancers. To study the role of Sei1, we have generated a mouse line deficient for this gene. Sei1-null fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects ...

  6. Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Pedersen, Mette Ø; Poulsen, Tim S; Gang, Anne O;

    2015-01-01

    Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number...

  7. Fertility potential after unilateral orchiopexy: an age independent risk of subsequent infertility when biopsies at surgery lack germ cells

    DEFF Research Database (Denmark)

    Cortes, Dina; Thorup, J M; Lindenberg, S

    1996-01-01

    We evaluated whether adult fertility potential was better when unilateral orchiopexy was done at ages 2 to 6 years or later, and we identified those at risk for infertility.......We evaluated whether adult fertility potential was better when unilateral orchiopexy was done at ages 2 to 6 years or later, and we identified those at risk for infertility....

  8. Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice.

    Science.gov (United States)

    De Stefano, M Egle; Leone, Lucia; Lombardi, Loredana; Paggi, Paola

    2005-12-01

    Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.

  9. Lactobacillus acidophilus CP23 with weak immunomodulatory activity lacks anchoring structure for surface layer protein.

    Science.gov (United States)

    Yanagihara, Sae; Kato, Shinji; Ashida, Nobuhisa; Yamamoto, Naoyuki

    2015-05-01

    To determine the reason for the low levels of Surface layer protein A (SlpA) on CP23 cells, which might play a crucial role in the immunomodulatory effect of Lactobacillus acidophilus, the DNA sequence of the slpA gene of CP23 and L-92 strains, including the upstream region, were analyzed. Unexpectedly, there was no significant difference in the predicted amino acid sequence of the C-terminus needed for cell anchoring, and only an additional Ala-Val-Ala sequence inserted in the N-terminal region of the mature CP23 protein. Therefore, anchoring of SlpA on the cell wall of CP23 and L-92 was evaluated by a reconstitution assay, which showed that SlpA released by LiCl treatment from both CP23 and L-92 was successfully anchored on LiCl-treated L-92 cells, but not on LiCl-treated CP23 cells. Moreover, quantitative analysis of SlpA protein in the culture medium of CP23 and L-92 by ELISA revealed higher levels of SlpA secretion in CP23 cells than in L-92 cells. Collectively, these results suggest that the lower levels of SlpA on the surface of CP23 cells might be caused by less cell wall capacity for SlpA anchoring, leading to an accumulation of SlpA in the culture medium of CP23 cells. The present study supports the importance of cell surface structure of L. acidophilus L-92 for SlpA anchoring on the cell surface needed for immunomodulatory effect.

  10. Cell density monitoring and control of microencapsulated CHO cell cultures

    OpenAIRE

    Cole, Harriet Emma

    2015-01-01

    Though mammalian cells play a key role in the manufacturing of recombinant glycosylated proteins, cell cultures and productivity are limited by the lack of suitable systems to enable stable perfusion culture. Microencapsulation, or entrapping cells within a semi-permeable membrane, offers the potential to generate high cell density cultures and improve the productivity by mimicking the cells natural environment. However, the cells being secluded by the microcapsules membrane are difficult to ...

  11. Assessment of NDE Methods to Detect Lack of Fusion in HDPE Butt Fusion Joints

    Energy Technology Data Exchange (ETDEWEB)

    Crawford, Susan L.; Doctor, Steven R.; Cinson, Anthony D.; Watts, Michael W.; Moran, Traci L.; Anderson, Michael T.

    2011-07-31

    Studies at the Pacific Northwest National Laboratory (PNNL) in Richland, Washington, were conducted to evaluate nondestructive examinations (NDE) coupled with mechanical testing of butt fusion joints in high-density polyethylene (HDPE) pipe for assessing lack of fusion. The work provided information to the United States Nuclear Regulatory Commission (NRC) on the effectiveness of volumetric inspection techniques of HDPE butt fusion joints in Section III, Division 1, Class 3, buried piping systems in nuclear power plants. This paper describes results from assessments using ultrasonic and microwave nondestructive techniques and mechanical testing with the high-speed tensile impact test and the side-bend test for determining joint integrity. A series of butt joints were fabricated in 3408, 12-inch (30.5-cm) IPS DR-11 HDPE material by varying the fusion parameters to create good joints and joints containing a range of lack-of-fusion conditions. Six of these butt joints were volumetrically examined with time-of-flight diffraction (TOFD), phased-array (PA) ultrasound, and the Evisive microwave system. The outer diameter (OD) weld beads were removed for microwave evaluation and the pipes ultrasonically re-evaluated. In two of the six pipes, both the outer and inner diameter (ID) weld beads were removed and the pipe joints re-evaluated. Some of the pipes were sectioned and the joints destructively evaluated with the high-speed tensile test and the side-bend test. The fusion parameters, nondestructive and destructive evaluation results have been correlated to validate the effectiveness of what each NDE technology detects and what each does not detect. There was no single NDE method that detected all of the lack-of-fusion flaws but a combination of NDE methods did detect most of the flaws.

  12. Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex.

    Science.gov (United States)

    Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-05-01

    The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

  13. Mechanics of cuticular elastic energy storage in leg joints lacking extensor muscles in arachnids.

    Science.gov (United States)

    Sensenig, Andrew T; Shultz, Jeffrey W

    2003-02-01

    Certain leg joints in arachnids lack extensor muscles and have elastically deformable transarticular sclerites spanning their arthrodial membranes, an arrangement consistent with a model in which flexor muscles load transarticular sclerites during flexion and energy from elastic recoil is used for extension. This study quantifies the potential contribution of elastic recoil to extension torque at joints of the fourth leg of representative arachnids. Extension torques of isolated joints with and without transarticular sclerites were measured as the joint was rotated through angles and at angular velocities comparable with those used by walking animals. The procedure was repeated with the joint subjected to different internal fluid pressures in order to assess the potential role of hydraulically induced extension. The efficiency of elastic energy storage (resilience) in the absence of internal fluid pressure was 70-90% for joints with well-developed transarticular sclerites, and the magnitude of torque was similar to those produced by different joint extension mechanisms in other arthropods. Increased internal fluid pressure acted synergistically with transarticular sclerites in some joints but had little or no effect in others. Joints that lacked both extensor muscles and transarticular sclerites appeared to be specialized for hydraulic extension, and joints operated by antagonistic muscles lacked apparent specializations for either elastic or hydraulic extension. It is well known that elastic energy storage is a significant contributor to propulsion in running vertebrates and certain arthropods, where elastic elements are loaded as the center of mass falls during one phase of the locomotor cycle. However, transarticular sclerites are apparently loaded by contraction of flexor muscles when the leg is not in contact with the substratum. Hence the mechanism of a transarticular sclerite is more similar to the flight and jumping mechanisms of other arthropods than to

  14. CONGENITAL CATARACT – LACK OF AWARENESS: EFFECTS ON DELAYED HOSPITALIZATION & TREAMENT

    Directory of Open Access Journals (Sweden)

    Anjaneyulu

    2015-01-01

    Full Text Available Study on congenital cataract conducted at regional eye hospital and in coordination with paediatric department. Histry, symptomatology and observations taken inconsideration within days , weeks , months after year. Daignosed by Torch Light examination and s lit lamp examination and B - SCAN examination. Very low number of cases detected out of large number of children screened . It may be due lack of awareness of parents to bring the children to the hospital and health personel. After diagnosing surgical interve ntion done. Post - operative visual prognosis good in some cases and some are amblyopic

  15. Lack of Arg972 polymorphism in the IRS1 gene in Parakanã Brazilian Indians.

    Science.gov (United States)

    Bezerra, Rosângela M N; Chadid, Thiago T; Altemani, Claúdia M; Sales, Teresa S I; Menezes, Raimundo; Soares, Manoel C P; Saad, Sara T O; Saad, Mario J A

    2004-02-01

    Several polymorphisms in the insulin receptor substrate-1 (IRS1) gene have been reported in the last years. The most common IRS1 variant, a Gly --> Arg substitution at codon 972 (Arg972 IRS1), is more prevalent among subjects who have features of insulin resistance syndrome associated, or not, with type 2 diabetes in European populations. To determine whether the absence of IRS1 polymorphism is a more general characteristic of Paleo-Indian-derived populations, we examined the Arg972 IRS1 polymorphism in Parakanã Indians and found a lack of this polymorphism in the Parakanã population.

  16. Reticulated acanthoma with sebaceous differentiation. Lack of association with Muir-Torre syndrome.

    Science.gov (United States)

    Haake, Dana L; Minni, John P; Nowak, Michael; Abenoza, Pascual; Nousari, Carlos H

    2009-06-01

    We hereby report a case of a reticulated acanthoma with sebaceous differentiation (RASD), a rare and often mislabeled benign lesion that is characterized by epidermal acanthosis and clusters of sebocytes in a reticulated seborrheic keratosis-like pattern. The presence of multiple sebaceous tumors, most notably cystic sebaceous adenomas and keratoacanthomas, has been associated with Muir-Torre syndrome (MTS). Although very rare, cases of RASD have been reported with MTS, which potentially offers profound clinical significance to this neoplasm. This case further supports the lack of association of MTS with RASD.

  17. Lack of shunt response in suspected idiopathic normal pressure hydrocephalus with Alzheimer disease pathology.

    Science.gov (United States)

    Hamilton, Roy; Patel, Sunil; Lee, Edward B; Jackson, Eric M; Lopinto, Joanna; Arnold, Steven E; Clark, Christopher M; Basil, Anuj; Shaw, Leslie M; Xie, Sharon X; Grady, M Sean; Trojanowski, John Q

    2010-10-01

    To determine the impact of cortical Alzheimer disease pathology on shunt responsiveness in individuals treated for idiopathic normal pressure hydrocephalus (iNPH), 37 patients clinically diagnosed with iNPH participated in a prospective study in which performance on neurologic, psychometric, and gait measures before and 4 months after shunting was correlated with amyloid β plaques, neuritic plaques, and neurofibrillary tangles observed in cortical biopsies obtained during shunt insertion. No complications resulted from biopsy acquisition. Moderate to severe pathology was associated with worse baseline cognitive performance and diminished postoperative improvement on NPH symptom severity scales, gait measures, and cognitive instruments compared to patients lacking pathology.

  18. Anti-NMDA receptor encephalitis associated with transient cerebral dyschromatopsia, prosopagnosia, and lack of stereopsis.

    Science.gov (United States)

    Sawamura, Hiromasa; Yamamoto, Tomotaka; Ohtomo, Ryo; Bannai, Taro; Wakakura, Masato; Tsuji, Shoji

    2014-06-01

    A 20-year-old woman suffered from anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis and was treated with removal of an ovarian teratoma and retroperitoneal ganglioneuroma in addition to immunotherapy. She was incapable of face recognition, had difficulty with object recognition, and lacked color sensation and stereo perception during recovery. These symptoms were transient and completely resolved over 4 months. Our report documents additional aspects of visual impairment associated with anti-NMDAR encephalitis and suggests that the disease can lead to diffuse cerebral dysfunction including the cortical visual system.

  19. Contribution to hospital performance: market orientation vs. marketing effort and lack of competition.

    Science.gov (United States)

    Wrenn, Bruce

    2002-01-01

    Marketing is still viewed with some skepticism by some hospital administrators who wonder if marketing is needed when the hospital is in a benign competitive environment. This research seeks to investigate the contribution of a marketing orientation to hospital performance beyond what can be achieved by merely spending money on promotion or not facing stiff competition. Findings reveal that having an authentic market orientation makes a significant contribution to a hospital's success above what can be achieved through promotional budgets and lack of competition.

  20. Analysis of complete mitochondrial genomes from extinct and extant rhinoceroses reveals lack of phylogenetic resolution

    DEFF Research Database (Denmark)

    Willerslev, Eske; Gilbert, Tom; Binladen, Jonas;

    2009-01-01

    genes is highly diffuse, with mixed topological support from different genes. Furthermore, the choice of outgroup (horse vs tapir) has considerable effect on reconstruction of the phylogeny. The lack of resolution is suggestive of a hard polytomy at the base of crown-group Rhinocerotidae......BACKGROUND: The scientific literature contains many examples where DNA sequence analyses have been used to provide definitive answers to phylogenetic problems that traditional (non-DNA based) approaches alone have failed to resolve. One notable example concerns the rhinoceroses, a group for which...

  1. Lack of the RNA chaperone hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Jakobsen, Henrik; Struve, Carsten;

    2012-01-01

    as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq...... is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host.......Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans...

  2. Lack of Buffering by Composites Promotes Shift to More Cariogenic Bacteria.

    Science.gov (United States)

    Nedeljkovic, I; De Munck, J; Slomka, V; Van Meerbeek, B; Teughels, W; Van Landuyt, K L

    2016-07-01

    Secondary caries (SC) remains a very important problem with composite restorations. The objectives of this study were to test the acid-buffering ability of several restorative materials and to evaluate whether buffering of the restorative material has an impact on the microbial composition of the biofilm. Disk-shaped specimens of conventional composite, composite with surface prereacted glass-ionomer filler particles (so-called giomer), glass-ionomer cement (GIC), amalgam, and hydroxyapatite (HAp) (control) were exposed to aqueous solutions with pH 4, 5, 6, and 7 and to the medium containing bacteria-produced acids, and pH changes were recorded over several days. Next, material specimens were immersed in bacterial growth medium with pH adjusted to 5. After a 24-h incubation, the extracts were collected and inoculated with a cariogenic (Streptococcus mutans) and a noncariogenic (Streptococcus sanguinis) species. The bacterial growth was monitored both in a single-species model by spectrophotometry and in a dual-species model by viability quantitative polymerase chain reaction. Amalgam and HAp showed the strongest acid-buffering ability, followed by the GIC and the giomer, while the conventional composite did not exhibit any buffering capacity. Furthermore, due to the lack of acid-buffering abilities, composite was not able to increase the pH of the medium (pH 5), which, in the absence of antibacterial properties, allowed the growth of S. mutans, while the growth of S. sanguinis, a less aciduric species, was completely inhibited. A similar effect was observed when bacteria were cultured together: there was a higher percentage of S. mutans and lower percentage of S. sanguinis with the conventional composite than with other materials and HAp. In conclusion, conventional composites lack the ability to increase the local pH, which leads to the outgrowth of more acidogenic/aciduric bacteria and higher cariogenicity of the biofilm. Together with lack of antibacterial

  3. The lack of theoretical support for using person trade-offs in QALY-type models

    DEFF Research Database (Denmark)

    Østerdal, Lars Peter Raahave

    2009-01-01

    Considerable support for the use of person trade-off methods to assess the quality-adjustment factor in quality-adjusted life years (QALY) models has been expressed in the literature. The WHO has occasionally used similar methods to assess the disability weights for calculation of disability......-adjusted life years (DALYs). This paper discusses the theoretical support for the use of person trade-offs in QALY-type measurement of (changes in) population health. It argues that measures of this type based on such quality-adjustment factors almost always violate the Pareto principle, and so lack normative...

  4. Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system

    OpenAIRE

    Narita, Yuka; Sato, Keiko; Yukitake, Hideharu; Shoji, Mikio; Nakane, Daisuke; Nagano, Keiji; Yoshimura, Fuminobu; Naito, Mariko; Nakayama, Koji

    2014-01-01

    Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, t...

  5. Skin Stem Cells in Skin Cell Therapy

    Directory of Open Access Journals (Sweden)

    Mollapour Sisakht

    2015-12-01

    Full Text Available Context Preclinical and clinical research has shown that stem cell therapy is a promising therapeutic option for many diseases. This article describes skin stem cells sources and their therapeutic applications. Evidence Acquisition Compared with conventional methods, cell therapy reduces the surgical burden for patients because it is simple and less time-consuming. Skin cell therapy has been developed for variety of diseases. By isolation of the skin stem cell from the niche, in vitro expansion and transplantation of cells offers a surprising healing capacity profile. Results Stem cells located in skin cells have shown interesting properties such as plasticity, transdifferentiation, and specificity. Mesenchymal cells of the dermis, hypodermis, and other sources are currently being investigated to promote regeneration. Conclusions Because skin stem cells are highly accessible from autologous sources and their immunological profile is unique, they are ideal for therapeutic approaches. Optimization of administrative routes requires more investigation own to the lack of a standard protocol.

  6. Increased contribution of L-arginine-nitric oxide pathway in aorta of mice lacking the gene for vimentin.

    Science.gov (United States)

    Zhang, J; Henrion, D; Ebrahimian, T; Benessiano, J; Colucci-Guyon, E; Langa, F; Lévy, B I; Boulanger, C M

    2001-10-01

    Experiments were designed to investigate endothelial function in the aorta of mice lacking the gene for the cytoskeleton protein vimentin (vim -/- ). Rings with and without endothelium from wild-type (vim +/+ ), heterozygous (vim +/- ), and homozygous (vim -/- ) mice were suspended in organ chambers to record of changes in isometric tension. During phenylephrine contraction, acetylcholine evoked comparable endothelium-dependent relaxations in the three groups. In the presence of Nomega-nitro-L-arginine, acetylcholine caused endothelium-dependent contractions, which were greater in vim -/- than in vim +/+ and vim +/- aortas. Indomethacin did not affect relaxation to acetylcholine in vim +/+ or in vim +/-, but it significantly increased the maximal response in vim -/- (67 +/- 7 vs. 102 +/- 4%). Response to acetylcholine in vim -/- aortas was not affected by cyclooxygenase type 2 inhibitor NS-398, the thromboxane receptor antagonist SQ-29,548, or superoxide dismutase. Relaxations to sodium nitroprusside were not different between vim +/+ and vim -/- mice and were not affected by cyclooxygenase inhibition. Cyclic guanosine monophosphate levels, which were increased to a comparable level by acetylcholine in vim +/+ and vim -/-, were augmented by indomethacin in vim -/- aortas but not in vim +/+ aortas. Expression of endothelial nitric oxide synthase was not different between vim +/+ and vim -/- preparations. These results suggest that despite comparable endothelium-dependent responses to acetylcholine, endothelial cells from vim -/- mice release a cyclooxygenase product that compensates the augmented contribution of nitric oxide.

  7. Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse.

    Science.gov (United States)

    Panicker, Sandip; Brown, Keith; Nicoll, Roger A

    2008-01-22

    Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

  8. Cellular resilience: 5-HT neurons in Tph2(-/-) mice retain normal firing behavior despite the lack of brain 5-HT.

    Science.gov (United States)

    Montalbano, Alberto; Waider, Jonas; Barbieri, Mario; Baytas, Ozan; Lesch, Klaus-Peter; Corradetti, Renato; Mlinar, Boris

    2015-11-01

    Considerable evidence links dysfunction of serotonin (5-hydroxytryptamine, 5-HT) transmission to neurodevelopmental and psychiatric disorders characterized by compromised "social" cognition and emotion regulation. It is well established that the brain 5-HT system is under autoregulatory control by its principal transmitter 5-HT via its effects on activity and expression of 5-HT system-related proteins. To examine whether 5-HT itself also has a crucial role in the acquisition and maintenance of characteristic rhythmic firing of 5-HT neurons, we compared their intrinsic electrophysiological properties in mice lacking brain 5-HT, i.e. tryptophan hydroxylase-2 null mice (Tph2(-/-)) and their littermates, Tph2(+/-) and Tph2(+/+), by using whole-cell patch-clamp recordings in a brainstem slice preparation and single unit recording in anesthetized animals. We report that the active properties of dorsal raphe nucleus (DRN) 5-HT neurons in vivo (firing rate magnitude and variability; the presence of spike doublets) and in vitro (firing in response to depolarizing current pulses; action potential shape) as well as the resting membrane potential remained essentially unchanged across Tph2 genotypes. However, there were subtle differences in subthreshold properties, most notably, an approximately 25% higher input conductance in Tph2(-/-) mice compared with Tph2(+/-) and Tph2(+/+) littermates (presilience to complete brain 5-HT deficiency.

  9. Mice lacking the transcriptional coactivator PGC-1α exhibit alterations in inhibitory synaptic transmission in the motor cortex.

    Science.gov (United States)

    Dougherty, S E; Bartley, A F; Lucas, E K; Hablitz, J J; Dobrunz, L E; Cowell, R M

    2014-06-20

    Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator known to regulate gene programs in a cell-specific manner in energy-demanding tissues, and its dysfunction has been implicated in numerous neurological and psychiatric disorders. Previous work from the Cowell laboratory indicates that PGC-1α is concentrated in inhibitory interneurons and is required for the expression of the calcium buffer parvalbumin (PV) in the cortex; however, the impact of PGC-1α deficiency on inhibitory neurotransmission in the motor cortex is not known. Here, we show that mice lacking PGC-1α exhibit increased amplitudes and decreased frequency of spontaneous inhibitory postsynaptic currents in layer V pyramidal neurons. Upon repetitive train stimulation at the gamma frequency, decreased GABA release is observed. Furthermore, PV-positive interneurons in PGC-1α -/- mice display reductions in intrinsic excitability and excitatory input without changes in gross interneuron morphology. Taken together, these data show that PGC-1α is required for normal inhibitory neurotransmission and cortical PV-positive interneuron function. Given the pronounced motor dysfunction in PGC-1α -/- mice and the essential role of PV-positive interneurons in maintenance of cortical excitatory:inhibitory balance, it is possible that deficiencies in PGC-1α expression could contribute to cortical hyperexcitability and motor abnormalities in multiple neurological disorders.

  10. Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism

    Science.gov (United States)

    Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

    2017-01-01

    Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis. PMID:28241048

  11. Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

    Science.gov (United States)

    Kiss, Krisztina; Csonka, Csaba; Pálóczi, János; Pipis, Judit; Görbe, Anikó; Kocsis, Gabriella F; Murlasits, Zsolt; Sárközy, Márta; Szűcs, Gergő; Holmes, Christopher P; Pan, Yijun; Bhandari, Ashok; Csont, Tamás; Shamloo, Mehrdad; Woodburn, Kathryn W; Ferdinandy, Péter; Bencsik, Péter

    2016-11-01

    Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, pEPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, pEPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents.

  12. Leukemia-associated NF1 inactivation in patients with pediatric T-ALL and AML lacking evidence for neurofibromatosis.

    Science.gov (United States)

    Balgobind, Brian V; Van Vlierberghe, Pieter; van den Ouweland, Ans M W; Beverloo, H Berna; Terlouw-Kromosoeto, Joan N R; van Wering, Elisabeth R; Reinhardt, Dirk; Horstmann, Martin; Kaspers, Gertjan J L; Pieters, Rob; Zwaan, C Michel; Van den Heuvel-Eibrink, Marry M; Meijerink, Jules P P

    2008-04-15

    Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder caused by mutations in the NF1 gene. Patients with NF1 have a higher risk to develop juvenile myelomonocytic leukemia (JMML) with a possible progression toward acute myeloid leukemia (AML). In an oligo array comparative genomic hybridization-based screening of 103 patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL) and 71 patients with MLL-rearranged AML, a recurrent cryptic deletion, del(17)(q11.2), was identified in 3 patients with T-ALL and 2 patients with MLL-rearranged AML. This deletion has previously been described as a microdeletion of the NF1 region in patients with NF1. However, our patients lacked clinical NF1 symptoms. Mutation analysis in 4 of these del(17)(q11.2)-positive patients revealed that mutations in the remaining NF1 allele were present in 3 patients, confirming its role as a tumor-suppressor gene in cancer. In addition, NF1 inactivation was confirmed at the RNA expression level in 3 patients tested. Since the NF1 protein is a negative regulator of the RAS pathway (RAS-GTPase activating protein), homozygous NF1 inactivation represent a novel type I mutation in pediatric MLL-rearranged AML and T-ALL with a predicted frequency that is less than 10%. NF1 inactivation may provide an additional proliferative signal toward the development of leukemia.

  13. Lack of Matrilin-2 favors liver tumor development via Erk1/2 and GSK-3β pathways in vivo.

    Directory of Open Access Journals (Sweden)

    Alexandra Fullár

    Full Text Available Matrilin-2 (Matn2 is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM. It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2(-/- mice comparing them with wild-type (WT mice in a diethylnitrosamine (DEN model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2(-/- mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2(-/- mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 μg/g body weight DEN treatment, the liver of Matn2(-/- mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3α/β and retinoblastoma protein (p-Rb, decrease in p21/CIP1 level, and increase in β-Catenin protein expression were detected in Matn2(-/- livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2(-/- livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3β, play strategic roles.

  14. Loneliness in patients with rheumatic diseases: the significance of invalidation and lack of social support.

    Science.gov (United States)

    Kool, Marianne B; Geenen, Rinie

    2012-01-01

    Rheumatic diseases affect about 20% of the population, leading to common symptoms such as joint problems, pain, fatigue, and stiffness. Loneliness is prevalent in individuals with rheumatic diseases. This could be due to not receiving social support and being stigmatized and invalidated, which might be most common in fibromyalgia, a rheumatic disease that lacks medical evidence. The aim of this study was to compare loneliness in distinct rheumatic diseases and to examine the association of loneliness with social support and invalidation. Participants were 927 patients with ankylosing spondylitis (n = 152), fibromyalgia (n = 341), osteoarthritis (n = 150), rheumatoid arthritis (n = 171), or systemic diseases (n = 113). They completed online questionnaires including an 11-point Likert scale assessing loneliness, the Illness Invalidation Inventory (3*1; Kool et al., 2010), and the Social Support Survey (SSS; De Boer, Wijker, Speelman, & De Haes, 1996; Sherbourne & Stewart, 1991). Patients with fibromyalgia experienced significantly more loneliness than patients with ankylosing spondylitis and patients with rheumatoid arthritis. Besides being younger, having lower education, and not working, in multiple regression analyses both lack of social support and invalidation were independently correlated with loneliness. This suggests that to decrease loneliness, therapeutic attention should be given to both increasing social support as well as decreasing invalidation in patients with rheumatic diseases, especially in patients with fibromyalgia.

  15. Migrant Workers’ Lack of Cultural Rights and Interests and Government Safeguard Measures

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    This paper analyses the status quo of migrant workers’ lack of cultural rights and interests as follows:the holistic cultural life of migrant workers is scant and monotonous;the cultural facilities provided by the majority of enterprises are critically short;the cultural life of migrant workers has the characteristic of closeness to some extent;the holistic cultural consuming capacity of migrant workers is very low,and they are not satisfied with their own cultural life.This paper puts forward the measures that the government should adopt to make up for the lack of migrant workers’ cultural rights and interests as follows:first,it should eliminate discrimination in terms of system,and incorporate migrant workers’ cultural rights and interests into the building scope of urban public culture;second,it should establish a variety of free public culture facilities,to adapt to the needs of migrant workers;third,it should formulate various kinds of laws and regulations,to effectively safeguard migrant workers’ cultural rights and interests.

  16. Marines, medics, and machismo: lack of fit with masculine occupational stereotypes discourages men's participation.

    Science.gov (United States)

    Peters, Kim; Ryan, Michelle K; Haslam, S Alexander

    2015-11-01

    Women have made substantial inroads into some traditionally masculine occupations (e.g., accounting, journalism) but not into others (e.g., military, surgery). Evidence suggests the latter group of occupations is characterized by hyper-masculine 'macho' stereotypes that are especially disadvantageous to women. Here, we explore whether such macho occupational stereotypes may be especially tenacious, not just because of their impact on women, but also because of their impact on men. We examined whether macho stereotypes associated with marine commandos and surgeons discourage men who feel that they are 'not man enough'. Study 1 demonstrates that male new recruits' (N = 218) perceived lack of fit with masculine commandos was associated with reduced occupational identification and motivation. Study 2 demonstrates that male surgical trainees' (N = 117) perceived lack of fit with masculine surgeons was associated with reduced identification and increased psychological exit a year later. Together, this suggests that macho occupational stereotypes may discourage the very men who may challenge them.

  17. Contamination cannot explain the lack of large-scale power in the cosmic microwave background radiation

    CERN Document Server

    Bunn, Emory F

    2008-01-01

    Several anomalies appear to be present in the large-angle cosmic microwave background (CMB) anisotropy maps of WMAP. One of these is a lack of large-scale power. Because the data otherwise match standard models extremely well, it is natural to consider perturbations of the standard model as possible explanations. We show that, as long as the source of the perturbation is statistically independent of the source of the primary CMB anisotropy, no such model can explain this large-scale power deficit. On the contrary, any such perturbation always reduces the probability of obtaining any given low value of large-scale power. We rigorously prove this result when the lack of large-scale power is quantified with a quadratic statistic, such as the quadrupole moment. When a statistic based on the integrated square of the correlation function is used instead, we present strong numerical evidence in support of the result. The result applies to models in which the geometry of spacetime is perturbed (e.g., an ellipsoidal U...

  18. Poor social performance of lonely people: lacking a skill or adopting a role?

    Science.gov (United States)

    Vitkus, J; Horowitz, L M

    1987-06-01

    A substantial literature has shown that lonely people differ from nonlonely people on a variety of measures of social performance. These differences have usually been conceptualized as a social skills deficit, which implies that lonely people lack the ability to perform appropriate and effective social behavior. Rather than a lack of this ability, the authors hypothesize that the adoption of passive interpersonal roles predisposes lonely people to exhibit inadequate performance. In order to test this hypothesis, lonely and nonlonely subjects were assigned to one of two roles: They either listened (Condition Li) to an interaction partner describe a personal problem or they themselves described a personal problem (Condition Pr) to their partner. The subjects' interpersonal role produced a substantial effect on their social behavior. Subjects who listened to their partner describe a problem generated more solutions to a set of hypothetical situations, attended to their partners more adequately, and conversed longer than did subjects who described a personal problem. In contrast, lonely subjects did not differ from nonlonely subjects in their social performance within each particular role. Lonely and nonlonely subjects did differ, however, in their subjective evaluations of themselves and of their performance. These results illustrate the need for research to address both the interpersonal and the intrapersonal bases of social performance.

  19. The importance of imagination (or lack thereof) in artificial, human and quantum decision making.

    Science.gov (United States)

    Gustafson, Karl

    2016-01-13

    Enlarging upon experiments and analysis that I did jointly some years ago, in which artificial (symbolic, neural-net and pattern) learning and generalization were compared with that of humans, I will emphasize the role of imagination (or lack thereof) in artificial, human and quantum cognition and decision-making processes. Then I will look in more detail at some of the 'engineering details' of its implementation (or lack thereof) in each of these settings. In other words, the question posed is: What is actually happening? For example, we previously found that humans overwhelmingly seek, create or imagine context in order to provide meaning when presented with abstract, apparently incomplete, contradictory or otherwise untenable decision-making situations. Humans are intolerant of contradiction and will greatly simplify to avoid it. They can partially correlate but do not average. Human learning is not Boolean. These and other human reasoning properties will then be taken to critique how well artificial intelligence methods and quantum mechanical modelling might compete with them in decision-making tasks within psychology and economics.

  20. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

    Directory of Open Access Journals (Sweden)

    Nir Qvit

    2016-04-01

    Full Text Available Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase and TRACK (Trypanosoma receptor for activated C-kinase. We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase, may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

  1. Compensatory evolution of gene regulation in response to stress by Escherichia coli lacking RpoS.

    Directory of Open Access Journals (Sweden)

    Daniel M Stoebel

    2009-10-01

    Full Text Available The RpoS sigma factor protein of Escherichia coli RNA polymerase is the master transcriptional regulator of physiological responses to a variety of stresses. This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition. This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they evolve in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than strains with functional RpoS. This phenotypic uniformity was caused by the same adaptive mutation in every independent population: the insertion of IS10 into the promoter of the otsBA operon. OtsA and OtsB are required to synthesize the osmoprotectant trehalose, and transcription of otsBA requires RpoS in the wild-type genetic background. The evolved IS10 insertion rewires expression of otsBA from RpoS-dependent to RpoS-independent, allowing for partial restoration of wild-type response to osmotic stress. Our results show that the regulatory networks of bacteria can evolve new structures in ways that are both rapid and repeatable.

  2. Behavioral characterization of a mutant mouse strain lacking D-amino acid oxidase activity.

    Science.gov (United States)

    Zhang, Min; Ballard, Michael E; Basso, Ana M; Bratcher, Natalie; Browman, Kaitlin E; Curzon, Pete; Konno, Ryuichi; Meyer, Axel H; Rueter, Lynne E

    2011-02-02

    D-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO(-)) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO-/- mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO-/- mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO-/- mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO-/- mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia.

  3. On the cell biology of pit cells, the liver-specific NK cells

    Institute of Scientific and Technical Information of China (English)

    Dian Zhong Luo; David Vermijlen; Bülent Ahishali; Vasilis Triantis; Georgia Plakoutsi; Filip Braet; Karin Vanderkerken; Eddie Wisse

    2000-01-01

    @@ INTRODUCTION Natural killer (NK) cells are functionally defined by their ability to kill certain tumor cells and virusinfected cells without prior sensitization[1]. NK cells comprise about 10% to 15% of lymphocytes in the peripheral blood and most of these cells in human and rat have the morphology of large granular lymphocytes ( LGL )[2]. However, recent studies have demonstrated that small agranular lymphocytes, lacking CD3 expression, have cytolytic activity comparable to NK cells[3].

  4. Accelerated tumor progression in mice lacking the ATP receptor P2X7.

    Science.gov (United States)

    Adinolfi, Elena; Capece, Marina; Franceschini, Alessia; Falzoni, Simonetta; Giuliani, Anna L; Rotondo, Alessandra; Sarti, Alba C; Bonora, Massimo; Syberg, Susanne; Corigliano, Domenica; Pinton, Paolo; Jorgensen, Niklas R; Abelli, Luigi; Emionite, Laura; Raffaghello, Lizzia; Pistoia, Vito; Di Virgilio, Francesco

    2015-02-15

    The ATP receptor P2X7 (P2X7R or P2RX7) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study, we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with postmortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intratumoral IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an antitumor immune response, and to restrict tumor growth and metastatic diffusion.

  5. Pancreatic ductal adenocarcinoma mice lacking mucin 1 have a profound defect in tumor growth and metastasis.

    Science.gov (United States)

    Besmer, Dahlia M; Curry, Jennifer M; Roy, Lopamudra D; Tinder, Teresa L; Sahraei, Mahnaz; Schettini, Jorge; Hwang, Sun-Il; Lee, Yong Y; Gendler, Sandra J; Mukherjee, Pinku

    2011-07-01

    MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.

  6. Lack of NF1 gene expression in a sporadic schwannoma from a patient without neurofibromatosis

    Energy Technology Data Exchange (ETDEWEB)

    Norton, K.K.; Dowton, B. [St. Louis Childrens Hospital, MO (United States); Silow-Santiago, I. [Washington Univ. School of Medicine, St. Louis, MO (United States)] [and others

    1994-09-01

    The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss of NF1 gene expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. We examined one spinal schwannoma from a patient without clinical features of neurofibromatosis type 1 or 2. The tumor was a typical schwannoma confirmed by standard neuropathologic criteria and expressed S100 by immunocytochemistry. NF1 gene expression in this tumor was examined by in situ hybridization using an NF1-specific riboprobe, Northern blot analysis and reverse-transcribed (RT) PCR. Little or no expression of NF1 RNA could be detected using these methods whereas abundant expression of S100, cyclophilin and beta-action RNA was found in the tumor. Fibroblast and Schwann cells were then individually cultured from this schwannoma and the RNA extracted for Northern blot and RT-PCR analysis. In these cultured Schwann cells both from early and late passages, abundant expression of NF1 RNA could be detected. It is unlikely that our culture technique preferentially expanded {open_quotes}normal{close_quotes} Schwann cells, since NF1 acts as a tumor suppressor gene and its presence would not confer any growth advantage over the tumor-derived, neurofibromin-negative Schwann cells which presumably have an increased proliferation rate. Similarly, the conditions used to expand these Schwann cells do not result in increased NF1 gene expression as shown in previous studies. These results suggest that, in some tumors, expression of the NF1 gene can be downregulated by factors produced within the tumor and that this type of tumor suppressor gene downregulation may represent another mechanism other than mutation for turning off the expression of these growth-suppressing genes and allowing for cell proliferation in tumors.

  7. Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations

    DEFF Research Database (Denmark)

    Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco;

    2016-01-01

    of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5(A214V)) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5...... patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue...

  8. Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2.

    Science.gov (United States)

    Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M S E; Ackermann, Teresa F; Föller, Michael; Schleicher, Erwin; Sheffield, William P; Arthur, J Simon C; Lang, Florian; Qadri, Syed M

    2015-11-27

    The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.

  9. Tanapoxvirus lacking a neuregulin-like gene regresses human melanoma tumors in nude mice.

    Science.gov (United States)

    Zhang, Tiantian; Suryawanshi, Yogesh R; Kordish, Dennis H; Woyczesczyk, Helene M; Jeng, David; Essani, Karim

    2017-02-01

    Neuregulin (NRG), an epidermal growth factor is known to promote the growth of various cell types, including human melanoma cells through ErbB family of tyrosine kinases receptors. Tanapoxvirus (TPV)-encoded protein TPV-15L, a functional mimic of NRG, also acts through ErbB receptors. Here, we show that the TPV-15L protein promotes melanoma proliferation. TPV recombinant generated by deleting the 15L gene (TPVΔ15L) showed replication ability similar to that of wild-type TPV (wtTPV) in owl monkey kidney cells, human lung fibroblast (WI-38) cells, and human melanoma (SK-MEL-3) cells. However, a TPV recombinant with both 15L and the thymidine kinase (TK) gene 66R ablated (TPVΔ15LΔ66R) replicated less efficiently compared to TPVΔ15L and the parental virus. TPVΔ15L exhibited more robust tumor regression in the melanoma-bearing nude mice compared to other TPV recombinants. Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma.

  10. Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate.

    Science.gov (United States)

    Jurkowska, Halina; Roman, Heather B; Hirschberger, Lawrence L; Sasakura, Kiyoshi; Nagano, Tetsuo; Hanaoka, Kenjiro; Krijt, Jakub; Stipanuk, Martha H

    2014-05-01

    The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.

  11. Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6.

    Science.gov (United States)

    Truksa, Jaroslav; Gelbart, Terri; Peng, Hongfan; Beutler, Ernest; Beutler, Bruce; Lee, Pauline

    2009-11-01

    Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo, we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.

  12. Lack of Population Structure in Coriander Populations Based on SDS (Seed Storage Protein Page Analysis

    Directory of Open Access Journals (Sweden)

    Gülsüm Yaldiz

    2016-08-01

    Full Text Available Genetic variation is prerequisite for plant breeding. Nothing information existed in the literature for available diversity of Coriander accession in Turkey. Plant breeding activities are negligible in Turkey. So in order to start effective plant breeding program in Turkey, information on the available genetic diversity is viable. Therefore we planned to study the genetic variation and population structure of 29 Coriander accessions by seed storage protein (SDS. SDS analysis elaborated the lack of population structure and genetic bottleneck in the Coriander accessions in Turkey. Based on the results of this study, it was clear that sampling strategy was not appropriate and plant introduction should be made from different sources and diverse genotypes should be used as parents to initialize the effective Turkish Coriander breeding program.

  13. Spirulina platensis Lacks Antitumor Effect against Solid Ehrlich Carcinoma in Female Mice

    Science.gov (United States)

    Barakat, Waleed; Elshazly, Shimaa M.; Mahmoud, Amr A. A.

    2015-01-01

    Spirulina is a blue-green alga used as a dietary supplement. It has been shown to possess anti-inflammatory, antioxidant, and hepatoprotective properties. This study was designed to evaluate the antitumor effect of spirulina (200 and 800 mg/kg) against a murine model of solid Ehrlich carcinoma compared to a standard chemotherapeutic drug, 5-fluorouracil (20 mg/kg). Untreated mice developed a palpable solid tumor after 13 days. Unlike fluorouracil, spirulina at the investigated two dose levels failed to exert any protective effect. In addition, spirulina did not potentiate the antitumor effect of fluorouracil when they were administered concurrently. Interestingly, their combined administration resulted in a dose-dependent increase in mortality. The present study demonstrates that spirulina lacks antitumor effect against this model of solid Ehrlich carcinoma and increased mortality when combined with fluorouracil. However, the implicated mechanism is still elusive. PMID:26366170

  14. Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3.

    Science.gov (United States)

    Perez-Alcazar, Marta; Daborg, Jonny; Stokowska, Anna; Wasling, Pontus; Björefeldt, Andreas; Kalm, Marie; Zetterberg, Henrik; Carlström, Karl E; Blomgren, Klas; Ekdahl, Christine T; Hanse, Eric; Pekna, Marcela

    2014-03-01

    Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.

  15. Altered response to benzodiazepine anxiolytics in mice lacking GABA B(1) receptors.

    Science.gov (United States)

    Mombereau, Cedric; Kaupmann, Klemens; van der Putten, Herman; Cryan, John F

    2004-08-16

    Recently, we demonstrated that mice lacking the GABA(B(1)) subunit were more anxious than wild-type animals in several behavioural paradigms, most notably in the light-dark test. In an attempt to assess the effects of classical benzodiazepine anxiolytics on anxiety-like behaviour observed in these mice, animals were administered either chlordiazepoxide (10 mg/kg, p.o.) or diazepam (7.5 mg/kg, p.o.) prior to testing in the light-dark box. Surprisingly, in contrast with the wild-type mice, neither benzodiazepines decreased anxiety-like behaviour in GABA(B(1))(-/-) mice. These data suggest that targeted deletion of GABA(B(1)) subunit alters GABA(A) receptor function in vivo.

  16. Spirulina platensis Lacks Antitumor Effect against Solid Ehrlich Carcinoma in Female Mice

    Directory of Open Access Journals (Sweden)

    Waleed Barakat

    2015-01-01

    Full Text Available Spirulina is a blue-green alga used as a dietary supplement. It has been shown to possess anti-inflammatory, antioxidant, and hepatoprotective properties. This study was designed to evaluate the antitumor effect of spirulina (200 and 800 mg/kg against a murine model of solid Ehrlich carcinoma compared to a standard chemotherapeutic drug, 5-fluorouracil (20 mg/kg. Untreated mice developed a palpable solid tumor after 13 days. Unlike fluorouracil, spirulina at the investigated two dose levels failed to exert any protective effect. In addition, spirulina did not potentiate the antitumor effect of fluorouracil when they were administered concurrently. Interestingly, their combined administration resulted in a dose-dependent increase in mortality. The present study demonstrates that spirulina lacks antitumor effect against this model of solid Ehrlich carcinoma and increased mortality when combined with fluorouracil. However, the implicated mechanism is still elusive.

  17. Spirulina platensis Lacks Antitumor Effect against Solid Ehrlich Carcinoma in Female Mice.

    Science.gov (United States)

    Barakat, Waleed; Elshazly, Shimaa M; Mahmoud, Amr A A

    2015-01-01

    Spirulina is a blue-green alga used as a dietary supplement. It has been shown to possess anti-inflammatory, antioxidant, and hepatoprotective properties. This study was designed to evaluate the antitumor effect of spirulina (200 and 800 mg/kg) against a murine model of solid Ehrlich carcinoma compared to a standard chemotherapeutic drug, 5-fluorouracil (20 mg/kg). Untreated mice developed a palpable solid tumor after 13 days. Unlike fluorouracil, spirulina at the investigated two dose levels failed to exert any protective effect. In addition, spirulina did not potentiate the antitumor effect of fluorouracil when they were administered concurrently. Interestingly, their combined administration resulted in a dose-dependent increase in mortality. The present study demonstrates that spirulina lacks antitumor effect against this model of solid Ehrlich carcinoma and increased mortality when combined with fluorouracil. However, the implicated mechanism is still elusive.

  18. Lack of founding Amerindian mitochondrial DNA lineages in extinct aborigines from Tierra del Fuego-Patagonia.

    Science.gov (United States)

    Lalueza, C; Pérez-Pérez, A; Prats, E; Cornudella, L; Turbón, D

    1997-01-01

    Ancient DNA from bones and teeth of 60 individuals from four extinct human populations from Tierra del Fuego-Patagonia (Selknam, Yamana, Kaweskar and Aonikenk) has been extracted and the mitochondrial DNA (mtDNA) amplified by using the polymerase chain reaction. High-resolution analysis of endonuclease restriction site variation in the mtDNA and sequencing of its hypervariable non-coding control region, revealed complete absence of two of the four primary mitochondrial haplotype groups present in contemporary Amerinds, namely A and B. In contrast, haplogroups C and D were found in all but one sample with frequencies of approximately 38% and 60%. These results, together with the decreasing incidence of group A in more southerly latitudes in the American continent and the absence of cluster B above 55 degrees North in America and Asia, argue that the first settlers entering America 21000-14000 years ago already lacked both mtDNA lineages.

  19. Lack-of-fit tests for assessing mean structures for continuous dose-response data

    DEFF Research Database (Denmark)

    Martinussen, Torben

    2011-01-01

    We review a range of lack-of-fit tests suitable for assessing the appropriateness of the mean function in dose-response models. The review encompasses both well-known tests and new tests based on recent developments in statistics, which we have extended to the dose-response case. We argue...... that the classical methods are inadequate in certain situations, where the new tests may be applied. Power comparisons are carried out by means of extensive simulation studies, covering both designs with and without replicates at small and large sample sizes. Three datasets from dose-response applications illustrate...... differences and similarities between the tests. The results suggest that the new tests perform better and exhibit a wider applicability....

  20. Testing Lack-of-fit for a Polynomial Errors-in-variables Model

    Institute of Scientific and Technical Information of China (English)

    Li-xing Zhu; Wei-xing Song; Heng-jian Gui

    2003-01-01

    When a regression model is applied as an approximation of underlying model of data, the model checking is important and relevant. In this paper, we investigate the lack-of-fit test for a polynomial errorin-variables model. As the ordinary residuals are biased when there exist measurement errors in covariables,we correct them and then construct a residual-based test of score type. The constructed test is asymptotically chi-squared under null hypotheses. Simulation study shows that the test can maintain the significance level well.The choice of weight functions involved in the test statistic and the related power study are also investigated.The application to two examples is illustrated. The approach can be readily extended to handle more general models.

  1. Walk-based measure of balance in signed networks: detecting lack of balance in social networks.

    Science.gov (United States)

    Estrada, Ernesto; Benzi, Michele

    2014-10-01

    There is a longstanding belief that in social networks with simultaneous friendly and hostile interactions (signed networks) there is a general tendency to a global balance. Balance represents a state of the network with a lack of contentious situations. Here we introduce a method to quantify the degree of balance of any signed (social) network. It accounts for the contribution of all signed cycles in the network and gives, in agreement with empirical evidence, more weight to the shorter cycles than to the longer ones. We found that, contrary to what is generally believed, many signed social networks, in particular very large directed online social networks, are in general very poorly balanced. We also show that unbalanced states can be changed by tuning the weights of the social interactions among the agents in the network.

  2. Lack of new antiinfective agents: Passing into the pre-antibiotic age?

    Institute of Scientific and Technical Information of China (English)

    Klaus; Brandenburg; Tobias; Schürholz

    2015-01-01

    The lack of newly developed antibiotics, together with the increase in multi-resistance of relevant pathogenic bacteria in the last decades, represents an alarming signal for human health care worldwide. The number of severely infected persons increases not only in developing but also in highly industrialized countries. This relates in first line to the most severe form of a bacterial infection, sepsis and the septic shock syndrome, with high mortality on critical care units. No particular anti-sepsis drug is available, and the therapy with conventional antibiotics more and more fails to provide a survival benefit. Due to the fact that the pharmaceutical industry has withdrawn to a high degree from the development of anti-infectious agents, a huge challenge for health care is approaching in the 21 st century. In this article, these problems are outlined and possible alternatives are presented which may be helpful to solve the problem.

  3. Some Properties of A Lack-of-Fit Test for a Linear Errors in Variables Model

    Institute of Scientific and Technical Information of China (English)

    Li-xing Zhu; Heng-jian Cui; K.W.Ng

    2004-01-01

    The relationship between the linear errors-in-variables model and the corresponding ordinary linear model in statistical inference is studied.It is shown that normality of the distribution of covariate is a necessary and su cient condition for the equivalence.Therefore,testing for lack-of-t in linear errors-in-variables model can be converted into testing for it in the corresponding ordinary linear model under normality assumption.A test of score type is constructed and the limiting chi-squared distribution is derived under the null hypothesis.Furthermore,we discuss the power of the test and the choice of the weight function involved in the test statistic.

  4. Lack of blinding of outcome assessors in animal model experiments implies risk of observer bias

    DEFF Research Database (Denmark)

    Bello, Segun; Krogsbøll, Lasse T; Gruber, Jan

    2014-01-01

    and caused by three pesticides experiments with very large observer bias, pooled ROR was 0.20 (95% CI, 0.07, 0.59) in contrast to the pooled ROR in the other seven experiments, 0.82 (95% CI, 0.57, 1.17). CONCLUSION: Lack of blinding of outcome assessors in animal model experiments with subjective outcomes......OBJECTIVES: To examine the impact of not blinding outcome assessors on estimates of intervention effects in animal experiments modeling human clinical conditions. STUDY DESIGN AND SETTING: We searched PubMed, Biosis, Google Scholar, and HighWire Press and included animal model experiments with both...... blinded and nonblinded outcome assessors. For each experiment, we calculated the ratio of odds ratios (ROR), that is, the odds ratio (OR) from nonblinded assessments relative to the corresponding OR from blinded assessments. We standardized the ORs according to the experimental hypothesis...

  5. Why are low-income teens more likely to lack health insurance than their younger peers?

    Science.gov (United States)

    Leininger, Lindsey Jeanne; Burns, Marguerite E

    2011-01-01

    Low-income teenagers are more likely to lack health insurance than younger children. Using data from the 2006, 2007, and 2008 rounds of the National Health Interview Survey, we examine whether differences between teens and younger children in socioeconomic factors, public health insurance eligibility, and observable family characteristics explain this apparent age-related coverage gap. Somewhat surprisingly, they do not. We find a highly robust age-coverage gradient among poor and near-poor children. Our results suggest the need to examine teen-specific insurance enrollment dynamics, particularly in families with no younger siblings, to optimize the effect of the newly enacted Patient Protection and Affordable Care Act on teens' insurance coverage.

  6. The lack of Financial Statement Spplication in Kosovo’s Farms and its Consequences

    Directory of Open Access Journals (Sweden)

    MSc. Jehona Shkodra

    2013-12-01

    Full Text Available The application of financial statement analysis in business activities is essential but this is often seen as frivolous in developing countries and in Kosovo as well . From the research conducted in hundred farms in different regions of Kosovo we understood that the application level of financial statement applied is low and in few farms it does not exist at all. The following research questions were used for this research: 1. Why does this situation happen? 2. To what extent are farmers familiar with the financial statement analysis? 3. What is the farmer’ willingness to advance in financial statement analysis field? 4. What would be the consequences due to the lack of application of financial statements?

  7. Mice lacking the PACAP type I receptor have impaired photic entrainment and negative masking

    DEFF Research Database (Denmark)

    Hannibal, J.; Brabet, P.; Fahrenkrug, J.

    2008-01-01

    The retinohypothalamic tract (RHT) is a retinofugal neuronal pathway which, in mammals, mediates nonimage-forming vision to various areas in the brain involved in circadian timing, masking behavior, and regulation of the pupillary light reflex. The RHT costores the two neurotransmitters glutamate...... and pituitary adenylate cyclase activating peptide (PACAP), which in a rather complex interplay are mediators of photic adjustment of the circadian system. To further characterize the role of PACAP/PACAP receptor type 1 (PAC1) receptor signaling in light entrainment of the clock and in negative masking behavior......, we extended previous studies in mice lacking the PAC1 receptor (PAC1 KO) by examining their phase response to single light pulses using Aschoff type II regime, their ability to entrain to non-24-h light-dark (LD) cycles and large phase shifts of the LD cycle (jet lag), as well as their negative...

  8. Telemedicine: The legal framework (or the lack of it) in Europe

    Science.gov (United States)

    Raposo, Vera Lúcia

    2016-01-01

    In the framework of European law telemedicine is, simultaneously, a health service and an information service, therefore, both regulations apply. In what concerns healthcare and the practice of medicine there are no uniform regulations at the European level. Concerning health services the most relevant achievement to regulate this domain is Directive 2011/24/EU. In what regards information and telecommunications we must have in consideration Directive 95/46/EU, Directive 2000/31/EC and Directive 2002/58/EC. However, many issues still lack uniform regulation, mainly the domain of medical liability and of medical leges artis. Probably such standardization will never take place, since the European Union does not have, until now, a common set of norms regarding tort and criminal liability, much less specific legal norms on medical liability. These gaps may jeopardize a truly European internal market in health services and hamper the development of telemedicine in the European zone. PMID:27579146

  9. Increased consumption of ethanol and sugar water in mice lacking the dopamine D2 long receptor.

    Science.gov (United States)

    Bulwa, Zachary B; Sharlin, Jordan A; Clark, Peter J; Bhattacharya, Tushar K; Kilby, Chessa N; Wang, Yanyan; Rhodes, Justin S

    2011-11-01

    Individual differences in dopamine D2 receptor (D2R) expression in the brain are thought to influence motivation and reinforcement for ethanol and other rewards. D2R exists in two isoforms, D2 long (D2LR) and D2 short (D2SR), produced by alternative splicing of the same gene. The relative contributions of D2LR versus D2SR to ethanol and sugar water drinking are not known. Genetic engineering was used to produce a line of knockout (KO) mice that lack D2LR and consequently have increased expression of D2SR. KO and wild-type (WT) mice of both sexes were tested for intake of 20% ethanol, 10% sugar water and plain tap water using established drinking-in-the-dark procedures. Mice were also tested for effects of the D2 antagonist eticlopride on intake of ethanol to determine whether KO responses were caused by lack of D2LR or overrepresentation of D2SR. Locomotor activity on running wheels and in cages without wheels was also measured for comparison. D2L KO mice drank significantly more ethanol than WT in both sexes. KO mice drank more sugar water than WT in females but not in males. Eticlopride dose dependently decreased ethanol intake in all groups except male KO. KO mice were less physically active than WT in cages with or without running wheels. Results suggest that overrepresentation of D2SR contributes to increased intake of ethanol in the KO mice. Decreasing wheel running and general levels of physical activity in the KO mice rules out the possibility that higher intake results from higher motor activity. Results extend the literature implicating altered expression of D2R in risk for addiction by delineating the contribution of individual D2R isoforms. These findings suggest that D2LR and D2SR play differential roles in consumption of alcohol and sugar rewards.

  10. Myocardial mitochondrial and contractile function are preserved in mice lacking adiponectin.

    Directory of Open Access Journals (Sweden)

    Martin Braun

    Full Text Available Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ-/- mice and wildtypes (WT. In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24% in ADQ-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions.

  11. Myocardial mitochondrial and contractile function are preserved in mice lacking adiponectin.

    Science.gov (United States)

    Braun, Martin; Hettinger, Niko; Koentges, Christoph; Pfeil, Katharina; Cimolai, Maria C; Hoffmann, Michael M; Osterholt, Moritz; Doenst, Torsten; Bode, Christoph; Bugger, Heiko

    2015-01-01

    Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ-/-) mice and wildtypes (WT). In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24%) in ADQ-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions.

  12. Predicting Effects of Ocean Acidification and Warming on Algae Lacking Carbon Concentrating Mechanisms.

    Directory of Open Access Journals (Sweden)

    Janet E Kübler

    Full Text Available Seaweeds that lack carbon-concentrating mechanisms are potentially inorganic carbon-limited under current air equilibrium conditions. To estimate effects of increased atmospheric carbon dioxide concentration and ocean acidification on photosynthetic rates, we modeled rates of photosynthesis in response to pCO2, temperature, and their interaction under limiting and saturating photon flux densities. We synthesized the available data for photosynthetic responses of red seaweeds lacking carbon-concentrating mechanisms to light and temperature. The model was parameterized with published data and known carbonate system dynamics. The model predicts that direction and magnitude of response to pCO2 and temperature, depend on photon flux density. At sub-saturating light intensities, photosynthetic rates are predicted to be low and respond positively to increasing pCO2, and negatively to increasing temperature. Consequently, pCO2 and temperature are predicted to interact antagonistically to influence photosynthetic rates at low PFD. The model predicts that pCO2 will have a much larger effect than temperature at sub-saturating light intensities. However, photosynthetic rates under low light will not increase proportionately as pCO2 in seawater continues to rise. In the range of light saturation (Ik, both CO2 and temperature have positive effects on photosynthetic rate and correspondingly strong predicted synergistic effects. At saturating light intensities, the response of photosynthetic rates to increasing pCO2 approaches linearity, but the model also predicts increased importance of thermal over pCO2 effects, with effects acting additively. Increasing boundary layer thickness decreased the effect of added pCO2 and, for very thick boundary layers, overwhelmed the effect of temperature on photosynthetic rates. The maximum photosynthetic rates of strictly CO2-using algae are low, so even large percentage increases in rates with climate change will not

  13. Getting what we pay for: innovations lacking in provider payment reform for chronic disease care.

    Science.gov (United States)

    Tynan, Ann; Draper, Debra A

    2008-06-01

    Despite wide recognition that existing physician and hospital payment methods used by health plans and other payers do not foster high-quality and efficient care for people with chronic conditions, little innovation in provider payment strategies is occurring, according to a new study by the Center for Studying Health System Change (HSC) commissioned by the California HealthCare Foundation. This is particularly disconcerting because the nation faces an increasing prevalence of chronic disease, resulting in continued escalation of related health care costs and diminished quality of life for more Americans. To date, most efforts to improve care of patients with chronic conditions have focused on paying vendors, such as disease management firms, to intervene with patients or redesigning care delivery without reforming underlying physician and hospital payment methods. While there is active discussion and anticipation of physician and hospital payment reform, current efforts are limited largely to experimental or small-scale pilot programs. More fundamental payment reform efforts in practice are virtually nonexistent. Existing payment systems, primarily fee for service, encourage a piecemeal approach to care delivery rather than a coordinated approach appropriate for patients with chronic conditions. While there is broad agreement that existing provider payment methods are not well aligned with optimal chronic disease care, there are significant barriers to reforming payment for chronic disease care, including: (1) fragmented care delivery; (2) lack of payment for non-physician providers and services supportive of chronic disease care; (3) potential for revenue reductions for some providers; and (4) lack of a viable reform champion. Absent such reform, however, efforts to improve the quality and efficiency of care for chronically ill patients are likely to be of limited success.

  14. Regional Fluctuation in the Functional Consequence of LINE-1 Insertion in the Mitf Gene: The Black Spotting Phenotype Arisen from the Mitfmi-bw Mouse Lacking Melanocytes.

    Directory of Open Access Journals (Sweden)

    Kazuhisa Takeda

    Full Text Available Microphthalmia-associated transcription factor (Mitf is a key regulator for differentiation of melanoblasts, precursors to melanocytes. The mouse homozygous for the black-eyed white (Mitfmi-bw allele is characterized by the white-coat color and deafness with black eyes due to the lack of melanocytes. The Mitfmi-bw allele carries LINE-1, a retrotransposable element, which results in the Mitf deficiency. Here, we have established the black spotting mouse that was spontaneously arisen from the homozygous Mitfmi-bw mouse lacking melanocytes. The black spotting mouse shows multiple black patches on the white coat, with age-related graying. Importantly, each black patch also contains hair follicles lacking melanocytes, whereas the white-coat area completely lacks melanocytes. RT-PCR analyses of the pigmented patches confirmed that the LINE-1 insertion is retained in the Mitf gene of the black spotting mouse, thereby excluding the possibility of the somatic reversion of the Mitfmi-bw allele. The immunohistochemical analysis revealed that the staining intensity for beta-catenin was noticeably lower in hair follicles lacking melanocytes of the homozygous Mitfmi-bw mouse and the black spotting mouse, compared to the control mouse. In contrast, the staining intensity for beta-catenin and cyclin D1 was higher in keratinocytes of the black spotting mouse, compared to keratinocytes of the control mouse and the Mitfmi-bw mouse. Moreover, the keratinocyte layer appears thicker in the Mitfmi-bw mouse, with the overexpression of Ki-67, a marker for cell proliferation. We also show that the presumptive black spots are formed by embryonic day 15.5. Thus, the black spotting mouse provides the unique model to explore the molecular basis for the survival and death of developing melanoblasts and melanocyte stem cells in the epidermis. These results indicate that follicular melanocytes are responsible for maintaining the epidermal homeostasis; namely, the present study

  15. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

    Directory of Open Access Journals (Sweden)

    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  16. The Impact of Lack of Resources on Declining Students' Enrolments in Design and Technology in Botswana Junior Secondary Schools

    Science.gov (United States)

    Gaotlhobogwe, Michael

    2012-01-01

    Lack of resources has resulted in declining students' enrolment in design and technology in Botswana junior secondary schools by up to 6% per year over 10 years, despite positive encouragement by the government. Based on the PATT (pupils' attitude towards technology) theoretical framework this study indicated how a lack of resources in Botswana…

  17. 37 CFR 1.489 - Protest to lack of unity of invention before the International Preliminary Examining Authority.

    Science.gov (United States)

    2010-07-01

    ... invention before the International Preliminary Examining Authority. 1.489 Section 1.489 Patents, Trademarks... Protest to lack of unity of invention before the International Preliminary Examining Authority. (a) If the applicant disagrees with the holding of lack of unity of invention by the International...

  18. Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response.

    Science.gov (United States)

    Hamilton, Holly M; Wilson, Ray; Blythe, Martin; Nehring, Ralf B; Fonville, Natalie C; Louis, Edward J; Rosenberg, Susan M

    2013-11-01

    Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind(-)) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA(-) SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.

  19. Lack of functioning intratumoral lymphatics in colon and pancreas cancer tissue.

    Science.gov (United States)

    Olszewski, Waldemar L; Stanczyk, Marek; Gewartowska, Magdalena; Domaszewska-Szostek, Anna; Durlik, Marek

    2012-09-01

    There are controversial views as to whether intratumoral or peritumoral lymphatics play a dominant role in the metastatic process. Most clinical observations originate from studies of colon cancer. Colon contains mucosa and submucosa rich in lymphatics and with high lymph formation rate. This seems to be a prerequisite for easy metastasis of cancer cells to regional lymph nodes. However, there are other tissues as pancreas with a rudimentary lymphatic network where cancer metastasis formation is as intensive as in colon cancer. This contradicts the common notion that intratumor lymphatics play major role in metastases. We visualized interstitial space and lymphatics in the central and peripheral regions of colon and pancreas tumors using the color stereoscopic lymphography and simultaneously immunohistochemical performed stainings specific for lymphatic and blood endothelial cells. The density of open and compressed lymphatic and blood vessels was measured in the tumor core and edge. There were very few lymphatics in the colon and pancreas tumor core but numerous minor fluid "lakes" with no visible connection to the peritumoral lymphatics. Lining of "lakes" did not express molecular markers specific for lymphatic endothelial cells. Dense connective tissue surrounding tumor foci did not contain lymphatics. Peritumoral lymphatics were irregularly distributed in both types of tumor and only sporadically contained cells that might be tumor cells. Similar lymphoscintigraphic and histological pictures were seen in colon and pancreas cancer despite of different structure of both tissues. This suggests a uniform reaction of tissues to the growing cancer irrespective of the affected organ.

  20. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    Science.gov (United States)

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.